title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 24hr 4sU - 2

description:
Library name: Ctrl_2_24h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 1hr 4sU - 2

status: Public on May 30 2025

type: SRA

city: Leuven

first: Sam

last: Bryce-Smith

address: Herestraat 49

department: VIB-KU Leuven Center for Brain and Disease Research

country: Belgium

institute: KU Leuven

description:
Library name: Ctrl_2_1h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP

library_source: transcriptomic

data_processing:
Using the ‘rna_seq_snakemake’ alignment pipeline ( https://github.com/frattalab/rna_seq_snakemake ), raw FASTQ files were quality trimmed using fastp with the parameter “qualified_quality_phred: 10”, and aligned without soft-clipping to the GRCh38 genome build using STAR (v2.7.0f) with gene models from GENCODE v34. GRAND-SLAM (v2.0.7b) was run on the aligned data using gene models from GENCODE v34 using the “-trim5p 10 -trim3p 10” parameter to ignore mismatches at the ends of reads.
Assembly: hg38
Supplementary files format and content: Tab-delimited file containing GRAND-SLAM 2.0 outputs for per-gene estimated new RNA fractions and number of reads with T-C conversions. For per-sample values, column names are space-separated, with the first field corresponding to the sample name. Each sample has the following columns: 'Readcount' (The total number of reads mapped to gene, non-normalised, multimapping reads counted fractionally), '0.05 quantile' (lower bound/5% quantile of the posterior distribution of the new-to-total RNA ratio for the gene), 'Mean' (mean of posterior distribution of new-to-total RNA ratio for the gene), 'MAP' (mode of posterior distribution of new-to-total RNA ratio for the gene), '0.95 quantile' (upper bound/95 % quantile of posterior distribution of new-to-total RNA ratio for the gene), 'alpha' (alpha parameter of the beta approximation of posterior distribution of new-to-total RNA ratio for the gene), 'beta' (beta parameter of the beta approximation of posterior distribution of new-to-total RNA ratio for the gene), 'Conversions' (total number of T-C conversions in this gene), 'Coverage' (total number of uridines covered by any reads), 'Double-Hits' (total number of conversions for gene, but only counting in positions where sequenced by both mates of paired-end reads ), 'Double-Hit Coverage' (total number of  uridines covered by any reads, counting in positions where sequenced by both mates of paired-end reads) and 'min2' (number of reads with at least 2 T-C mismatches). The table has 3 'standard' columns: 'Gene' (Ensembl gene ID), 'Symbol' (the gene symbol/name) and 'Length' (length of longest transcript of gene in nt).
Library strategy: SLAM-seq

molecule: total RNA

organism: Homo sapiens

extract_protocol:
RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher).
Iodoacetamide (IAA) treatment was applied to each sample, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2 x  250 bp) on an Illumina NovaSeq SP.

tag: cell line

tag: cell type

value: iPSC-derived cortical neuron


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 12hr 4sU - 1

description:
Library name: Ctrl_1_12h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 24hr 4sU - 1

description:
Library name: Ctrl_1_24h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 8hr 4sU - 1

description:
Library name: Ctrl_1_8h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: TSB933_BJ_HPGA_anti-p65_RW+TPCA500

city: La Jolla

first: Tom

last: Suter

state: CA

address: 9500 Gilman Dr

department: Department of Medicine

country: USA

institute: UCSD

library_source: genomic

data_processing:
Data was aligned to hg18 using STAR-aligner with parameters adjusted avoid spliced alignments.
Aligned reads were processed for downstream analysis using the HOMER (Heinz et al., 2010) “makeTagDirectory” program, filtering for tags that were uniquely mapped and only selecting at most one sequencing read per position per length of read.
bigWig files were generated using the makeBigWig.pl function in HOMER, using the -chipseq style parameter
Genome_build: hg18
Supplementary_files_format_and_content: .bigWig : display of ChIP-seq data on genome browser as a graph

molecule: genomic DNA

source_name: Foreskin dermal fibroblast

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:4 8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.  Cells grown in rapamycin were introduced to and maintained in 10nM rapamycin after recovery from thawing.  Rapamycin maintained cells were collected for RNA-seq and ChIP-seq at PD31 and PD66 to correspond with similar PD young and senescent non-rapamycin treated cells.  For rapamycin withdrawal experiments, rapamycin maintained cells were grown to arrest, which typically occurred between PD75-85.  These cells are termed High Passage Growth Arrested.  At this point, the cells were divided into two plates that were either maintained or withdrawn from rapamycin for 2 weeks until they were collected.

extract_protocol:
Fixed chromatin immunoprecipitation assays were performed as previously described (Li et al., 2013), with the following modifications: Samples prepared for ChIP of histone modifications were fixed in 1% formaldehyde for 10 minutes at room temperature, while samples prepared for ChIP of transcription factors were double cross-linked in either 2mM DSG alone for 45 minutes at room temperature followed by 10 minute fixation with 1% formaldehyde.  After neutralization with 0.125M glycine, samples underwent nuclear extraction, and were afterwards resuspended in nuclear lysis buffer (50mM Tris/HCl pH7.4, 1% SDS, 10mM EDTA, 1x dissolved cOmplete Protease Inhibitor Cocktail (Sigma 11836145001)), and samples were sonicated using the Bioruptor (Diagenode).  The concentration of chromatin in each sample was detected using the Qubit Fluorometer (Thermo Fisher), and samples were then diluted 1:10 in dilution buffer (20mM Tris/HCl pH7.4, 100mM NaCl, 0.5% Triton-X-100, 2mM EDTA, 1x dissolved cOmplete Protease Inhibitor Cocktail).  Between 20-50 ug of chromatin from each sample was then used for each histone or transcription factor ChIP assay, respectively, and the volume of each sample was raised to 0.5 mL using 1:10 sonication:dilution buffer mixture.  Antibodies were then incubated with each sample overnight.    Antibody:chromatin complexes were then immunoprecipitated as described using 20 uL Protein G Dynabeads (Thermo Fisher), which were then washed using the following washes:  ChIPs of histone modifications were washed with the following buffers in order: 2xWB1 (20mM Tris/HCl pH7.4, 150mM NaCl, 1% Triton-X-100, 0.1% SDS, 2mM EDTA), 1xWB2 (20mM Tris/HCl pH7.4, 500mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB3 (10mM Tris/HCl pH7.4, 250mM LiCl, 1% NP-40, 1% Deoxycholate, 1mM EDTA), 2xTE (10mM Tris/HCl pH 8.0, 1mM EDTA).  ChIPs of transcription factors were washed with the following buffers in order: 2xWB1 (20mM Tris/HCl pH7.4, 150mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB2 (20mM Tris/HCl pH7.4, 300mM NaCl, 1% Triton-X-100, 2mM EDTA), 1xWB3 (20mM Tris/HCl pH7.4, 400mM NaCl, 1% Triton-X-100, 2mM EDTA), 2xTE (10mM Tris/HCl pH 8.0, 1mM EDTA).  After washing, samples were eluted overnight at 65 °C as described, purified using the QIAquick PCR Purification Kit (QIAGEN), and were eluted in EB (1% SDS, 0.1M NaHCO3).  Samples were then further sonicated for 10 minutes to ensure the majority of purified chromatin fell between 100-300bp.
Purified chromatin was then prepared for deep sequencing with the Illumina HiSeq 2500 system using either the KAPA LTP Library Preparation Kit (KK8232) or KAPA Hyper Prep Kit (KK8500) for histone or transcription factor ChIPs, respectively, following manufacturer’s instructions.  Samples were run on the Illumina HiSeq 2500 as single-read reactions of 50 cycles.

treatment_protocol: High Passage Growth Arrested BJ fibroblasts maintained in 10nM rapamycin were then either maintained or withdrawn from rapamycin for 14 additional days, and treated with either control or the IKK/p65 inhibitor TPCA-1 (500nM) during this 14 day period.

value: BJ Fibroblast

tag: antibody

value: p65:Santa Cruz sc-372 (C-20)

tag: Stage

value: High Passage Growth Arrested

tag: treatment

value: Rapamycin Withdrawn + TPCA


title: Subj.PNMC311_Day0

type: RNA

city: Palo Alto

first: Dmitri

middle: A

last: Kazmin

address: 240 Pasteur Dr

department: Institute for Immunity, Transplantation and Infection

institute: Stanford University

hyb_protocol: Samples were hybridized on  HG-133-plus-PM plates

scan_protocol: Plates were scanned on GeneTitan multichannel instrument (Affymetrix)

data_processing: Probe level processing was done by RMA with log2 transform and probe summary by median polish

label: Biotin

source_name: PBMC

label_protocol: Fragemented cRNA was lebeled with biotin

extract_protocol: RNA was isolated from whole blood Paxgene tubes using RNAeasy kit (Quagen)

tag: subject

tag: time point


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 4hr 4sU - 1

description:
Library name: Ctrl_1_4h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: Subj.PNMB231_Day7


title: TSB931_BJ_HPGA_anti-p65_RM

value: Rapamycin Maintained


title: Subj.PNMC314_Day0


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 1hr 4sU - 1

description:
Library name: Ctrl_1_1h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: Subj.PNMB231_Day0


title: TSB919_BJ_HPGA_anti-H3K27ac_RW+TPCA500

value: H3K27ac:abcam ab4729


title: SLAM-seq of Homo sapiens: iPSC-derived cortical neurons (i3Neurons) with TDP-43 knockdown - control 0hr 4sU - 1

description:
Library name: Ctrl_1_0h
Human iPS cells used in this study were engineered from WTC11 line to express mouse or human neurogenin-2 under a doxycycline-inducible promoter (Fernandopulle et al., Current Protocols in Cell Biology, 2018; Wang et al., Stem Cell Reports, 2017) and an enzymatically dead Cas9 (+/- CAG-dCas9-BFP-KRAB) at safe-harbour loci (Tian et al., Neuron, 2019). CRISPRi knockdown of TDP-43 in iPSCs was achieved using sgRNA targeting the transcription start site of TARDBP (GGGAAGTCAGCCGTGAGACC) or a non-targeting control (GTCCACCCTTATCTAGGCTA), delivered by lentiviral transduction. Cortical-like i3Neurons were induced as previously described (Fernandopulle et al., Current Protocols in Cell Biology, 2018). SLAM-sequencing was performed on cortical-like i3Neurons following protocols adapted from Herzog et al., Nature Methods, 2017. Samples were treated with 100  M 4sU on Day 7 for 0, 1, 4, 8, 12, 24 hours before immediate wash with phosphate buffered saline (PBS). RNA was extracted using the Qiagen RNA isolation and purification kit. RNA concentration was estimated using a Nanodrop Microvolume Spectrophotometer (Thermofisher). After ensuring an adequate amount of RNA in each sample, iodoacetamide (IAA) treatment was applied to each, facilitating the thiol modification of incorporated 4sU. Sequencing libraries were prepared with Kapa RiboErase RNA Hyper kit and sequenced (2   250 bp) on an Illumina NovaSeq SP


title: Subj.PNMB229_Day7


title: TSB932_BJ_HPGA_anti-p65_RW

value: Rapamycin Withdrawn


title: TSB918_BJ_HPGA_anti-H3K27ac_RW


title: Subj.PNMB229_Day0


title: Subj.PNMB226_Day7


title: TSB917_BJ_HPGA_anti-H3K27ac_RM


title: HBEC WGBS, WTSal2

city: Davis

first: Hong

last: Ji

address: 1 Shields Ave

department: California National Primate Research Center

institute: University of California, Davis

description:
Library name: Control2
WTSal2

data_processing:
We used the CpG_Me pipeline to process and align the WGBS data. Because of methylation biases at the 5’ and 3’ ends of reads, we trimmed 10 bases from the 3’ end of both reads, and 10 and 20 bases from the 5’ of read 1 and read 2 respectively using Trim Galore
Bismark was then used to align the trimmed reads to the hg38 human reference genome, and to generate CpG count matrices which were used in downstream analyses
The reads were aligned to the human transcriptome (hg38) using Bowtie2
MultiQC was used for quality control of the fastq data and the alignments to the reference genome
Following the above data processing, differentially methylated regions (DMRs) were identified for three different comparisons: 1) KD saline vs. WT saline, 2) WT HDM vs. WT saline, and 3) KD HDM vs. WT HDM. We used DMRichR to perform these differential methylation analyses. For our differential methylation analyses, we required at least 1x coverage for a CpG for all samples included in a given comparison, a minimum of 5 CpGs for a DMR, 10 permutations for DMR and block analyses, and the single CpG coefficient required to discover testable background regions to be at least 0.05. For a region to be considered a DMR, we required an empirical p-value ≤ 0.01 and a methylation difference of at least 10%.
Assembly: hg38
Supplementary files format and content: tab-delimited text files include methylation percentage for each cytosine included in the analysis, one file per sample

source_name: Human bronchial epithelial cells

growth_protocol: HBECs were grown in 1X K-SFM supplemented with EGF, pituitary extract, and pen-strep (complete media). A 12 well plate was seeded with cultured HBEC with near confluent numbers and incubated overnight for attachment. After 24 hours, the complete media was replaced with 1X K-SFM with no serum supplements (minimal media) and incubated for an additional 8 hours.

extract_protocol:
DNA was extracted using the Zymo DNA Clean and Concentrator-5 Kit
Whole genome bisulfite sequencing (WGBS) libraries were prepared for a total of 8 samples (2 each from these samples groups: WT saline, KD saline, WT HDM, and KD HDM) using Swift’s Accel-NGS Methyl-Seq Kit. We checked the quality of the libraries using an Agilent 2100 Bioanalyzer, and we measured library concentration using a Qubit high sensitivity DNA assay

treatment_protocol: TET1 was knocked down by adding 30 pmol/mL siRNA (ThermoFisher) and incubated for 24 hours in minimal media. Following control siRNA or TET1 siRNA incubation, saline or house dust mite (HDM, Greer) was added to HBEC at a concentration of 25ug/mL and incubated for another 24 hours before collection.

value: Human bronchial epithelial cells

tag: genotype

value: Wildtype

value: Saline


title: HBEC WGBS, TET1KDHDM1

description:
Library name: KDHDM1
KDHDM1

value: TET1 knockdown

value: House dust mite


title: HBEC WGBS, TET1KDSal1

description:
Library name: KD1
KDSal1


title: HBEC WGBS, WTSal1

description:
Library name: Control1
WTSal1


title: HBEC WGBS, TET1KDSal2

description:
Library name: KD2
KDSal2


title: TSB907_BJ_HPGA_anti_H3K4me2_RM

value: H3K4me2:abcam ab7766


title: TSB908_BJ_HPGA_anti_H3K4me2_RW


title: HBEC WGBS, WTHDM2

description:
Library name: HDM2
WTHDM2


title: HBEC WGBS, TET1KDHDM2

description:
Library name: KDHDM2
KDHDM2


title: HBEC WGBS, WTHDM1

description:
Library name: HDM1
WTHDM1


title: HBEC RNA-seq, TET1KDHDM1

description:
Library name: KDHDM1
KDHDM1

data_processing:
Read quality was assessed using FastQC
Reads were dynamically trimmed using Trim Galore with the following settings: bases with less than a quality score of 20 were trimmed, 10 bases were trimmed from the 5’ end of both read 1 and read 2, the adapter stringency was set to 6, and reads smaller than 50 bases after trimming were discarded
The reads were aligned to the human transcriptome (hg38) using Bowtie2
Transcripts were subsequently quantified using RSEM
The data were then converted into DESeq2 format using tximport
We used DESeq2 for differential expression analyses
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes DESeq2 normalized counts for each Sample

molecule: polyA RNA

extract_protocol:
RNA (1µg per sample) was submitted for poly-A RNA library preparation at Novogene (Sacramento, CA)
RNA (1µg per sample) was submitted for poly-A RNA library preparation and sequencing at Novogene (Sacramento, CA)


title: HBEC RNA-seq, TET1KDHDM2

description:
Library name: KDHDM2
KDHDM2


title: TSB722_BJ_HPGA_RW_Rep2

data_processing:
Data was aligned to hg18 using STAR-aligner, with parameters adjusted to account for spliced alignment.
Aligned reads were processed for downstream analysis using the HOMER (Heinz et al., 2010) “makeTagDirectory” program, filtering for tags that were uniquely mapped and only selecting at most one sequencing read per position per length of read.
bigWig files were generated using the makeBigWig.pl function in HOMER, using the -rnaseq style parameter.
Genome_build: hg18
Supplementary_files_format_and_content: .bigWig : display of ChIP-seq data on genome browser as a graph

extract_protocol:
Total cellular RNA was harvested from cells using the RNeasy Mini Kit (QIAGEN-74104), making use of the optional DNase treatment step.
Samples were then prepared for sequencing with the Illumina HiSeq 2500 system using the TruSeq Stranded Total RNA LT Sample Prep Kit (RS-122-2201/RS-122-2202) for ribosomal RNA depleted libraries.  Samples were run on the Illumina HiSeq 2500 as single-read reactions of 50 cycles.

treatment_protocol: High Passage Growth Arrested BJ fibroblasts that were maintained in 10nM rapamycin were split into 2 separate wells that were either maintained in or withdrawn from 10nM rapamycin treatment.


title: HBEC RNA-seq, WTHDM2

description:
Library name: HDM2
WTHDM2


title: Subj.PNMB224_Day7


title: TSB721_BJ_HPGA_RM_Rep2

value: Rapamycin Withdrawal


title: Subj.PNMC308_Day7


title: HBEC RNA-seq, TET1KDSal4

description:
Library name: KD4
KDSal4


title: HBEC RNA-seq, TET1KDSal3

description:
Library name: KD3
KDSal3


title: TSC742_BJ_HPGA_RW_Rep1


title: Subj.PNMC308_Day0


title: Subj.PNMB224_Day0


title: Subj.PNMB226_Day0


title: Subj.PNMB219_Day0


title: HBEC RNA-seq, TET1KDSal1

description:
Library name: KD1
KDSal1


title: HBEC RNA-seq, WTSal4

description:
Library name: Control4
WTSal4


title: Subj.PNMB219_Day7


title: HBEC RNA-seq, WTSal3

description:
Library name: Control3
WTSal3


title: TSF10_BJ_Senescent_Inhibin+TGFB2_Rep2

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:4 8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.

extract_protocol:
Total cellular RNA was harvested from cells using the RNeasy Mini Kit (QIAGEN-74104), making use of the optional DNase treatment step.
Samples were then prepared for sequencing with the Illumina HiSeq 2500 system using the TruSeq Stranded Total RNA LT Sample Prep Kit (RS-122-2201/RS-122-2202) for ribosomal RNA depleted libraries.  Samples were run on the Illumina HiSeq 2500 as single-read reactions of 75 cycles.

treatment_protocol: SMAD2/3 regulating ligands Activin A (100ng/ml), TGFB2 (1ng/ml), Inhibin (100ng/ml)+TGFB2 (1ng/ml), or Inhibin (100ng/ml) were given to senescent (>PD60) BJ fibroblasts for 2 weeks, after which cells were harvested for RNA-seq.

value: Senescent

value: Inhibin + Tgfb2


title: TSF9_BJ_Senescent_Inhibin+TGFB2_Rep1


title: HBEC ATAC, WTHDM2

description:
Library name: HDM2
WTHDM2

data_processing:
Reads were mapped to hg38 using BowTie2 and peaks were called using MACS2
Based on sample clustering and other QC metrics, two samples from each sample group were selected for further downstream analysis
Principle component analysis and unsupervised clustering were used to evaluate data quality
Differential peaks were identified using MANorm2. We identified differentially accessible peaks (FDR ≤ 0.05, fold-change ≥ 2) in three different comparisons: 1) KD saline vs. WT saline, 2) WT HDM vs. WT saline, and 3) KD HDM vs. WT HDM
Assembly: hg38
Supplementary files format and content: bigwig files with chromatin accessibility information for each sample

extract_protocol:
DNA was extracted using the Zymo DNA Clean and Concentrator-5 Kit
ATAC-seq libraries were prepared for a total of 12 samples (initial n = 3 for WT saline, n = 3 for WT HDM, n = 3 for KD saline, HDM, n = 3 for KD HDM). Samples were prepared according to the Omni-ATAC-seq protocol. Briefly, 50,000 viable cells were lysed and nuclei were extracted. Nuclei were then treated with transposase at 37°C for 30 minutes (Nx#-tde1, tagment DNA enzyme, Illumina). DNA was extracted using (Zymo DNA Clean and Concentrator-5 Kit) and libraries were prepared. Preamplification with 5 cycles was performed and qPCR was used to determine additional PCR cycles. Libraries were then cleaned up and their quality was determined by bioanalyzer. Indexed libraries were then mixed and sequenced at the DNA technologies core at UC Davis across two sequencing runs


title: TSF8_BJ_Senescent_TGFB2_Rep2


title: HBEC ATAC, TET1KDHDM1

description:
Library name: KDHDM1
KDHDM1


title: Subj.PNMB218_Day0


title: HBEC RNA-seq, TET1KDSal2

description:
Library name: KD2
KDSal2


title: HBEC ATAC, TET1KDSal2

description:
Library name: KD2
KDSal2


title: HBEC ATAC, WTHDM1

description:
Library name: HDM1
WTHDM1


title: Subj.PNMB215_Day7


title: TSF7_BJ_Senescent_TGFB2_Rep1


title: HBEC RNA-seq, WTSal1

description:
Library name: Control1
WTSal1


title: Subj.PNMB214_Day7


title: Subj.PNMB218_Day7


title: Subj.PNMB215_Day0


title: HBEC RNA-seq, WTHDM1

description:
Library name: HDM1
WTHDM1


title: HBEC ATAC, TET1KDSal1

description:
Library name: KD1
KDSal1


title: Subj.PNMA123_Day7


title: TSF6_BJ_Senescent_Inhibin_Rep2

value: Inhibin


title: BM3, repeat 2

city: Sapporo

first: Jintao

last: He

state: Hokkaido

address: N15, W7, Kita-ku

department: Department of Cancer Pathology

country: Japan

institute: HOKKAIDO UNIVERSITY

description: Library name: H1975_BM3_2

data_processing:
After obtaining the FASTQ files, the data were cleaned using fastp. Using GRCh38 as the reference genome, the Salmon pipeline generated Count and TPM (Transcripts Per Kilobase Million) matrices for further analysis.
Assembly: GRCh38
Supplementary files format and content: CSV files include TPM values for each Sample

extract_protocol:
RNA was extracted from four cell lines (H1975, H1975-BM1, H1975-BM2, and H1975-BM3) following the standard protocol of the RNeasy Mini Kit (Qiagen). Three independent samples were prepared for each cell line.
The RNA samples were then sent to BGI’s Japan branch for sequencing on the DNBSEQ platform (PE150, 6G data per sample). All samples were sequenced simultaneously to avoid batch effects.

tag: source cell_type

value: EGFR-mutant lung adenocarcinoma cell line


title: HBEC ATAC, WTSal2

description:
Library name: Control2
WTSal2


title: TSF5_BJ_Senescent_Inhibin_Rep1

value: BJ Fibroblast


title: BM3, repeat 1

description: Library name: H1975_BM3_1


title: TSF4_BJ_Senescent_Activin_Rep2

value: Activin A


title: HBEC RNA-seq, WTSal2

description:
Library name: Control2
WTSal2


title: HBEC ATAC, WTSal1

description:
Library name: Control1
WTSal1


title: TSF3_BJ_Senescent_Activin_Rep1


title: BM3, repeat 3

description: Library name: H1975_BM3_3


title: BM2, repeat 2

description: Library name: H1975_BM2_2


title: Subj.PNMA123_Day0


title: TSF2_BJ_Senescent_Control_Rep2

value: DMSO


title: Subj.PNMC307_Day0


title: Subj.PNMB211_Day7


title: BM2, repeat 1

description: Library name: H1975_BM2_1


title: Subj.PNMB214_Day0


title: TSF1_BJ_Senescent_Control_Rep1


title: HEK293T, No Centrifugation, Rep3, Ribo-Seq

city: Cork

first: Gary

last: Loughran

address: Bioinnovation Hub, FSB 330, College Rd,

country: Ireland

institute: EIRNA Bio

data_processing:
The adaptor sequence of the ribo-seq and 1st read of rna-seq is AGATCGGAAGAGCACACGTCTGAACTCCAGTCA and the adaptor of the 2nd read of the rnaseq is AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT. These along with   first 3 nucleotides of each read was removed using Cutadapt. For the riboseq data the last 8 nucleotides of each reads remaining after removal of adaptor sequence consisted of the UMI sequence. This sequence was removed and used to de-multiplex the data. After removal of reads mapping to rRNA, tRNA and snRNA the remaining reads were aligned to a modified version Gencode41 transcriptome using Bowtie
Assembly: hg38
Supplementary files format and content: Tab-delineated raw count files. Reads are weighted by the number of locations to which the mapped, for example a unambiguously mapped read is worth 1 while a read mapping to two genes contributes 0.5 to each gene.
Library strategy: Ribo-Seq

growth_protocol: HEK293T and Huh-7 cells were maintained in DMEM supplemented with 10% foetal bovine serum, 1 mM L-glutamine and penicillin-streptomycin, and cultured at 37°C and 5% CO2.

extract_protocol:
Cells were lysed with ice-cold polysome lysis buffer (20 mM Tris-Cl pH 7.5, 150 mM NaCl, 5 mM MgCl2, in nuclease-free water, supplemented with 1 mM DTT, 0.1 mg/ml CHX, 25 U/ml Turbo DNase, and either 1%, 0.5%, or 0.1% Triton X-100) in triplicate. Lysates with centrifugation were clarified at 4°C for 5 mins at 10,000 x g. For Ribo-Seq, lysates of 15µg total RNA were treated with 35U RNase I (Biosearch Technologies) at 23°C for 1 hour at 400 rpm. Following T4 PNK end repair, ribosome-protected fragments were size-selected between 28-30nt via 15% TBE-urea polyacrylamide gel electrophoresis. Contaminating rRNA fragments were hybridised to a custom pool of 5’-biotinylated oligos with subsequent removal of these duplexes via affinity to streptavidin beads. A pre-adenylated linker bearing a unique molecular identifiers (UMI) was ligated to footprints. For RNA-seq, total RNA was extracted from lysates of 5µg RNA using TRIzol Reagent (Invitrogen) according to manufacturer's instructions.
For Ribo-Seq, cDNA synthesis was carried out using the SMARTer smRNA-Seq Kit for Illumina (Takara Bio) according to manufacturer's instructions, excluding polyadenylation and using a custom reverse transcription primer instead of the 3’ smRNA dT Primer provided, then amplified by 12-14 rounds of PCR. For RNA-seq, library construction was carried out with rRNA depletion using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions.
Ribo-Seq, RNA-seq

value: embyonic kidney cells

value: WT

value: No Centrifugation


title: Subj.PNMC307_Day7


title: BM1, repeat 3

description: Library name: H1975_BM1_3


title: BM1, repeat 2

description: Library name: H1975_BM1_2


title: HEK293T, No Centrifugation, Rep3, RNA-seq

data_processing:
The adaptor sequence of the ribo-seq and 1st read of rna-seq is AGATCGGAAGAGCACACGTCTGAACTCCAGTCA and the adaptor of the 2nd read of the rnaseq is AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT. These along with   first 3 nucleotides of each read was removed using Cutadapt. For the riboseq data the last 8 nucleotides of each reads remaining after removal of adaptor sequence consisted of the UMI sequence. This sequence was removed and used to de-multiplex the data. After removal of reads mapping to rRNA, tRNA and snRNA the remaining reads were aligned to a modified version Gencode41 transcriptome using Bowtie
Assembly: hg38
Supplementary files format and content: Tab-delineated raw count files. Reads are weighted by the number of locations to which the mapped, for example a unambiguously mapped read is worth 1 while a read mapping to two genes contributes 0.5 to each gene.


title: TSF40_BJ_HPGA_RM_TGFB2_Rep2

treatment_protocol: High Passage Growth Arrested BJ fibroblasts maintained in 10nM rapamycin were split into 2 separate wells and were either maintained in or withdrawn from 10nM rapamycin treatment for 14 days, and were treated with SMAD2/3 regulating ligands Activin A (100ng/ml), TGFB2 (1ng/ml), Inhibin (100ng/ml)+TGFB2 (1ng/ml), or Inhibin (100ng/ml) for those 14 days.

value: High Passage Growth Arrested

value: Rapamycin Maintained + Tgfb2


title: TSF42_BJ_HPGA_RW_TGFB2_Rep2

value: Rapamycin Withdrawal + Tgfb2


title: Subj.PNMB211_Day0


title: HEK293T, No Centrifugation, Rep1, Ribo-Seq


title: HEK293T, No Centrifugation, Rep2, Ribo-Seq


title: TSF41_BJ_HPGA_RW_TGFB2_Rep1


title: Subj.PNMC305_Day7


title: Primary, repeat 3

description: Library name: H1975_3


title: TSC741_BJ_HPGA_RM_Rep1

value: Rapamycin Maintained


title: TSF39_BJ_HPGA_RM_TGFB2_Rep1

value: Rapamycin Maintained+ Tgfb2


title: HEK293T, Centrifugation, Rep3, Ribo-Seq


title: HEK293T, No Centrifugation, Rep2, RNA-seq


title: HEK293T, Centrifugation, Rep2, Ribo-Seq

value: Centrifugation


title: BM1, repeat 1

description: Library name: H1975_BM1_1


title: TSF37_BJ_HPGA_RW_ActivinA_Rep1

value: Rapamycin Withdrawal + Activin A


title: TSF38_BJ_HPGA_RW_ActivinA_Rep2


title: Primary, repeat 2

description: Library name: H1975_2


title: HEK293T, No Centrifugation, Rep1, RNA-seq


title: Subj.PNMB210_Day0


title: Subj.PNMB210_Day7


title: Primary, repeat 1

description: Library name: H1975_1


title: Subj.PNMA121_Day7


title: BM2, repeat 3

description: Library name: H1975_BM2_3


title: TSF36_BJ_HPGA_RM_ActivinA_Rep2

value: Rapamycin Maintained + Activin A


title: Huh7, 1% Triton, Rep3, Pellet, RNA-seq

value: hepatocellular carcinoma cells


title: Huh7, 1% Triton, Rep3, Supernatant, RNA-seq

value: 1% Triton


title: Subj.PNMC305_Day0


title: MLO-Y4 cells, Hypoxia (2% O₂) for 24 hours, hypoxia_1

city: Sendai

first: Kohei

last: Narita

state: Miyagi

address: 4-1 Seiryo-machi, Aoba-ku

department: Dentistry

institute: Tohoku University

description:
Library name: hypoxia_1
Transcriptome analysis of murine MLO-Y4 osteocyte-like cells cultured under hypoxic conditions (2% O₂) for 24 hours

data_processing:
FastQC (v0.12.0) was used to assess read quality.Adapter sequences and low-quality reads were trimmed using DRAGEN RNA pipeline (v3.8.4).
Reads were aligned to the mouse reference genome (GRCm39, RefSeq mm39) using the DRAGEN RNA pipeline. Transcript quantification was performed at both gene and transcript levels (TPM values).
DEG analysis was conducted using the DRAGEN Differential Expression tool (v4.0.3). A threshold of |log2 fold change| ≥1 and adjusted p-value < 0.05 was applied to identify significantly differentially expressed genes.
Assembly: mm39
Supplementary files format and content: xlsx file containing transcript-level expression values generated from RNA-seq data. Each row represents a transcript or gene, and columns contain expression values such as TPM and counts across different sample groups. The file includes results from differential expression analyses comparing hypoxia and control conditions.

organism: Mus musculus

source_name: bone

growth_protocol: MLO-Y4 osteocytic cells were cultured in α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). Cells were maintained at 37°C under 5% CO₂

extract_protocol:
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. To remove residual genomic DNA, RNA samples were treated with RNase-Free DNase Set (Qiagen). RNA quality was assessed using an Agilent 2100 Bioanalyzer, and only samples with an RNA Integrity Number (RIN) ≥8 were used for library preparation.
RNA libraries for RNA-seq were prepared using the Illumina Stranded mRNA Prep, Ligation Kit  following manufacturer’s protocol

treatment_protocol: For hypoxia treatment, MLO-Y4 cells were cultured under hypoxic (2% O₂) conditions for 24 hours using a hypoxic chamber (BIONIX). Control samples were maintained under normoxic conditions(20% O₂) .

tag: tissue

value: bone

value: Osteocyte-like cells

value: Hypoxia (2% O₂) for 24 hours


title: TSF22_BJ_HPGA_RW_Inhibin+TGFB2_Rep2

value: BJ Fibroblast

value: Rapamycin Withdrawal + Inhibin + Tgfb2


title: Subj.PNMA119_Day7


title: MLO-Y4 cells, Normoxia (20% O₂) for 24 hours, control_4

description:
Library name: control_4
Transcriptome analysis of murine MLO-Y4 osteocyte-like cells cultured under normoxic conditions (20% O₂) for 26 hours.

value: WT

value: Normoxia (20% O₂) for 24 hours


title: TSF21_BJ_HPGA_RW_Inhibin+TGFB2_Rep1

value: High Passage Growth Arrested


title: MLO-Y4 cells, Hypoxia (2% O₂) for 24 hours, hypoxia_4

description:
Library name: hypoxia_4
Transcriptome analysis of murine MLO-Y4 osteocyte-like cells cultured under hypoxic conditions (2% O₂) for 26 hours


title: Huh7, 1% Triton, Rep3, Ribo-Seq


title: Subj.PNMC304_Day7


title: Subj.PNMA121_Day0


title: HEK293T, Centrifugation, Rep1, Ribo-Seq

value: embyonic kidney cells


title: Subj.PNMA119_Day0


title: Huh7, 1% Triton, Rep2, Pellet, RNA-seq


title: Huh7, 1% Triton, Rep2, Supernatant, RNA-seq


title: MLO-Y4 cells, Hypoxia (2% O₂) for 24 hours, hypoxia_2

description:
Library name: hypoxia_2
Transcriptome analysis of murine MLO-Y4 osteocyte-like cells cultured under hypoxic conditions (2% O₂) for 25 hours


title: Huh7, 1% Triton, Rep2, Ribo-Seq


title: MLO-Y4 cells, Normoxia (20% O₂) for 24 hours, control_3

description:
Library name: control_3
Transcriptome analysis of murine MLO-Y4 osteocyte-like cells cultured under normoxic conditions (20% O₂) for 25 hours.


title: MLO-Y4 cells, Normoxia (20% O₂) for 24 hours, control_2

description:
Library name: control_2
Transcriptome analysis of murine MLO-Y4 osteocyte-like cells cultured under normoxic conditions (20% O₂) for 24 hours.


title: TSF35_BJ_HPGA_RM_ActivinA_Rep1


title: Huh7, 1% Triton, Rep1, Pellet, RNA-seq


title: TSF18_BJ_HPGA_RW_Inhibin_Rep2

value: Rapamycin Withdrawal + Inhibin


title: Huh7, 1% Triton, Rep1, Supernatant, RNA-seq


title: TSF17_BJ_HPGA_RW_Inhibin_Rep1


title: TSF20_BJ_HPGA_RM_Inhibin+TGFB2_Rep2

value: Rapamycin Maintained + Inhibin + Tgfb2


title: Osteoclasr precursor cells, Dexamethasone-5

city: Jinan

first: Tao

last: Yuan

address: Central District: No. 324, Jingwu Weiqi Road, Huaiyin District

country: China

institute: Shandong University

description:
Library name: Dex5
Dex5

data_processing:
The sequencing quality were assessed with FastQC (v0.11.5) and then low quality data were filtered using NGSQC (v2.3.3).The clean reads were then aligned to the reference genome using HISAT2 (v2.1.0) with default parameters. The processed reads from each sample were aligned using HISAT2 against the reference genome. The gene expression analyses were performed with StringTie (v1.3.3b). DESeq(v1.28.0) was used to analyze the DEGs between samples. Thousands of independent statistical hypothesis testing was conducted on DEGs, separately. Then a p-value was obtained, which was corrected by FDR method. And Corrected P-value (q-value) was calculated by correcting using BH method. p-value or q-value were used to conduct significance  analysis. Parameters for classifying significantly are ≥2-fold differences (|log2FC|≥1, FC: the fold change of expressions) in the transcript abundance and p ≤ 0.05.
Assembly: The genome of the mouse reference genome (GRCm39) was used as reference.
Supplementary files format and content: genes.rawcount.anno.xlsx

source_name: bone marrow

growth_protocol: Bone marrow cells were isolated from femurs and tibias of six-month-old male mice, and cultured in complete medium (α-MEM with 10% FBS and 1% P/S) for 16–24 h. Then non-adherent bone marrow-derived macrophages (BMMs) were collected, centrifuged, resuspended, and then incubated into complete medium containing 10ng/ml M-CSF in a density of 2×105 cells/ml for 3 days. After that, BMMs were incubated in osteoclast-induction medium (complete medium containing 10ng/ml M-CSF and 25ng/ml RANKL). BMMs differentiated into osteoclast precursor cells (OPCs) after 24 hours induction. Followed each induction, Dex (10-8M) was applied to mimic GIONFH environment in vitro,

extract_protocol:
Total RNA was extracted with AG RNAex Pro Reagent (AG21102, Accurate Biotechnology) and assessed with Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) and Qubit Fluorometer (Invitrogen). Total RNA samples that meet the following requirements were used in subsequent experiments: RNA integrity number (RIN) > 7.0 and a 28S:18S ratio > 1.8.
RNA-seq libraries were generated and sequenced by CapitalBio Technology (Beijing, China). The triplicate samples of all assays were constructed an independent library, and do the following sequencing and analysis. The NEB Next Ultra RNA Library Prep Kit for Illumina (NEB) was used to construct the libraries for sequencing. NEB Next Poly(A) mRNA Magnetic Isolation Module (NEB) kit was used to enrich the poly(A) tailed mRNA molecules from 1 μg total RNA. The mRNA was fragmented into ~200 base pair pieces. The first-strand cDNA was synthesized from the mRNA fragments reverse transcriptase and random hexamer primers, and then the second-strand cDNA was synthesized using DNA polymerase I and RNaseH. The end of the cDNA fragment was subjected to an end repair process that included the addition of a single “A” base, followed by ligation of the adapters. Products were purified and enriched by polymerase chain reaction (PCR) to amplify the library DNA. The final libraries were quantified using KAPA Library Quantification kit (KAPA Biosystems, South Africa) and an Agilent 2100 Bioanalyzer. After quantitative reverse transcription-polymerase chain reaction (RT-qPCR) validation, libraries were subjected to paired-end sequencing with pair end 150-base pair reading length on an Illumina NovaSeq sequencer (Illumina).

treatment_protocol: OPCs treated with RANKL (25ng/ml) for 24 hours were control group, while OPCs co-treated with RANKL (25ng/ml) and Dex (10-8M) for 24 hours were Dex group.

value: bone marrow

value: primary cells

value: osteoclast precurosr cells

value: RANKL and Dexamethasone stimulation

tag: batch


title: Osteoclasr precursor cells, Dexamethasone-4

description:
Library name: Dex4
Dex4


title: Subj.PNMA116_Day0


title: Osteoclasr precursor cells, Dexamethasone-3

description:
Library name: Dex3
Dex3


title: TSF16_BJ_HPGA_RM_Inhibin_Rep2

value: Rapamycin Maintained + Inhibin


title: Huh7, 0.5% Triton, Rep3, Pellet, RNA-seq

value: hepatocellular carcinoma cells

value: 0.5% Triton


title: TSF15_BJ_HPGA_RM_Inhibin_Rep1


title: TSF19_BJ_HPGA_RM_Inhibin+TGFB2_Rep1


title: Subj.PNMC302_Day0


title: Subj.PNMC304_Day0


title: Osteoclasr precursor cells, Dexamethasone-6

description:
Library name: Dex6
Dex6


title: Subj.PNMB207_Day0


title: Osteoclasr precursor cells, Dexamethasone-2

description:
Library name: Dex2
Dex2


title: Huh7, 0.5% Triton, Rep3, Supernatant, RNA-seq


title: Osteoclasr precursor cells, Dexamethasone-1

description:
Library name: Dex1
Dex1


title: TSF33_BJ_HPGA_RW_Control_Rep3

value: Rapamycin Withdrawal


title: TSF34_BJ_HPGA_RW_Control_Rep4


title: Subj.PNMC302_Day7


title: Osteoclasr precursor cells, RANKL-6

description:
Library name: C6
C6

value: RANKL stimulation


title: Huh7, 0.5% Triton, Rep3, Ribo-Seq


title: TSF32_BJ_HPGA_RM_Control_Rep4

value: BJ Fibroblast

value: Rapamycin Maintained


title: Huh7, 1% Triton, Rep1, Ribo-Seq

value: WT

value: 1% Triton


title: Huh7, 0.5% Triton, Rep2, Pellet, RNA-seq


title: Osteoclasr precursor cells, RANKL-5

description:
Library name: C5
C5


title: TSF31_BJ_HPGA_RM_Control_Rep3

value: High Passage Growth Arrested


title: Osteoclasr precursor cells, RANKL-4

description:
Library name: C4
C4


title: Huh7, 0.5% Triton, Rep2, Supernatant, RNA-seq


title: Subj.PNMA111_Day7


title: Subj.PNMA113_Day0


title: Huh7, 0.5% Triton, Rep2, Ribo-Seq


title: Osteoclasr precursor cells, RANKL-3

description:
Library name: C3
C3


title: Subj.PNMB207_Day7


title: TSF14_BJ_HPGA_RW_Control_Rep2


title: Subj.PNMA111_Day0


title: Subj.PNMA113_Day7


title: TSF13_BJ_HPGA_RW_Control_Rep1


title: Huh7, 0.5% Triton, Rep1, Pellet, RNA-seq


title: Osteoclasr precursor cells, RANKL-2

description:
Library name: C2
C2


title: TSF12_BJ_HPGA_RM_Control_Rep2


title: Huh7, 0.5% Triton, Rep1, Supernatant, RNA-seq


title: Osteoclasr precursor cells, RANKL-1

description:
Library name: C1
C1


title: Huh7, 0.5% Triton, Rep1, Ribo-Seq


title: TSF11_BJ_HPGA_RM_Control_Rep1


title: Huh7, 0.1% Triton, Rep3, Pellet, RNA-seq

value: 0.1% Triton


title: HBEC ATAC, TET1KDHDM2

description:
Library name: KDHDM2
KDHDM2

value: Human bronchial epithelial cells

value: TET1 knockdown

value: House dust mite


title: TSC310_BJ_Senescent_shSMAD3-72_Rep2

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.

treatment_protocol: Lentivirus was produced through transfection of packaging plasmids into 293T (ATCC® CRL-3216) cells grown to ~80% confluency.  All transfections were done using Lipofectamine 2000 (Thermo Fisher 12566014), following the manufacturer’s protocol, with the ratios of plasmids and Lipofectamine 2000 reagent listed below.  Lentivirus for use in shRNA knockdown experiments was generated by transfecting a 0.625 : 1.875 : 2.5 ug ratio of MDC envelope plasmid: psPAX2 packaging plasmid: pLKO.1 shRNA expressing transfer plasmid with 17ul Lipofectamine 2000 in a 6-well plate well.  Media was changed 6 hours post transfection, and the viral containing media was collected and replaced at 48 and 72 hours post transfection.  Viral containing media was centrifuged at 600g for 5 min and the viral supernatant was collected and stored at 4°C prior to transduction.  Lentiviral transduction was performed on senescent BJ fibroblasts at ~50% confluency.   pLKO.1 stable knockdown lines were generated using a 48 ul : 1952 ul ratio of pLKO.1 viral supernatant with fresh culture media.  All transduction mixtures were given to cells and allowed to incubate overnight with polybrene added at a final concentration of 8ug/ml.  Knockdown cells were then maintained in 0.6ug/ml puromycin starting 4 days after transduction, and cells were harvested 14 days after transduction.

value: Senescent

tag: shRNA

value: shRNA - SMAD3 - Construct 72


title: Spleen_MPA_Rep5

city: Atlanta

first: Chris

last: Scharer

state: GA

address: 1510 Clifton Rd, Suite 3086A

department: Microbiology and Immunology

institute: Emory University

description: Library name: KAR032_S47

data_processing:
Raw sequencing reads were mapped to the mm10 version of the mouse genome using STAR and the UCSC mm10 known gene reference transcriptome.
Reads mapping to exons were summarized for each unique ENTREZ gene using the GenomicRanges package in R/Bioconductor.
Assembly: mm10
Supplementary files format and content: txt file of RPKM normalized read counts for each detected gene

source_name: Spleen

extract_protocol:
Cells were FACS sorted into RLT + 1%BME and snap frozen using the indicated markers. Total RNA was purified using the Quick-RNA MicroPrep Kit (Zymo Research)
Purified RNA was used as input for the SMART-seq v4 cDNA synthesis kit (Takara) and used as an input for the NexteraXT kit with the NexteraXT indexing primers (Illumina).

value: Spleen

tag: Sex

value: Female

value: NK cell (Live lymphocytes, CD45.2+, DX5+, NK1.1+, Ly6G-, CD3-, CD19-)

value: MPA


title: Huh7, 0.1% Triton, Rep3, Supernatant, RNA-seq

value: hepatocellular carcinoma cells


title: Subj.PNMA116_Day7


title: Subj.PNMB205_Day0


title: Subj.PNMB204_Day7


title: TSC309_BJ_Senescent_shSMAD2-71_Rep2

value: shRNA - SMAD2 - Construct 71


title: Spleen_MPA_Rep3

description: Library name: KAR030_S45


title: Huh7, 0.1% Triton, Rep3, Ribo-Seq


title: Subj.PNMB204_Day0


title: Spleen_MPA_Rep2

description: Library name: KAR029_S44


title: TSC301_BJ_Senescent_shGFPO_Rep2

value: BJ Fibroblast

value: shRNA - GFP - Control


title: Huh7, 0.1% Triton, Rep2, Pellet, RNA-seq

value: WT


title: TSC106_BJ_Senescent_shNFIX_64_Rep1

value: shRNA - NFIX - Construct 64


title: Spleen_MPA_Rep1

description: Library name: KAR028_S43


title: TSC105_BJ_Senescent_shNFIC_100_Rep1

value: shRNA - NFIC - Construct 100


title: Huh7, 0.1% Triton, Rep2, Supernatant, RNA-seq

value: 0.1% Triton


title: Subj.PNMA109_Day0


title: Subj.PNMB205_Day7


title: TSC104_BJ_Senescent_shNFIB_69_Rep1

value: shRNA - NFIB - Construct 69


title: Spleen_MPA_Rep4

description: Library name: KAR031_S46


title: Subj.PNMA109_Day7


title: Subj.PNMA107_Day0


title: Subj.PNMA107_Day7


title: Huh7, 0.1% Triton, Rep2, Ribo-Seq


title: Spleen_D8_Rep5

description: Library name: KAR027_S42

value: Pseudopregnancy


title: Huh7, 0.1% Triton, Rep1, Pellet, RNA-seq


title: Subj.PNMA105_Day0


title: Spleen_D8_Rep4

description: Library name: KAR026_S41


title: TSC102_BJ_Senescent_shp65-1_Rep1

value: shRNA - p65 - Construct 1


title: Spleen_D8_Rep2

description: Library name: KAR024_S39


title: Subj.PNMA105_Day7


title: Huh7, 0.1% Triton, Rep1, Ribo-Seq


title: Huh7, 0.1% Triton, Rep1, Supernatant, RNA-seq


title: TSC101_BJ_Senescent_shGFP_Rep1

value: Senescent


title: TSC103_BJ_Senescent_shNFIA_21_Rep1

value: shRNA - NFIA - Construct 21


title: Spleen_D8_Rep3

description: Library name: KAR025_S40

value: Spleen


title: MPA_UFRT_pospos_Rep4

description: Library name: KAR015_S30

source_name: Upper reproductive tract

value: Upper reproductive tract

value: Female


title: Spleen_D8_Rep1

description: Library name: KAR023_S38

value: NK cell (Live lymphocytes, CD45.2+, DX5+, NK1.1+, Ly6G-, CD3-, CD19-)


title: TSC422_BJ_HPGA_RW_shGFPN_Rep4

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:4 8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.  Cells grown in rapamycin were introduced to and maintained in 10nM rapamycin after recovery from thawing.  For rapamycin withdrawal experiments, rapamycin maintained cells were grown to arrest, which typically occurred between PD75-85.  These cells are termed High Passage Growth Arrested.

treatment_protocol: Lentivirus was produced through transfection of packaging plasmids into 293T (ATCC® CRL-3216) cells grown to ~80% confluency.  All transfections were done using Lipofectamine 2000 (Thermo Fisher 12566014), following the manufacturer’s protocol, with the ratios of plasmids and Lipofectamine 2000 reagent listed below.  Lentivirus for use in shRNA knockdown experiments was generated by transfecting a 0.625 : 1.875 : 2.5 ug ratio of MDC envelope plasmid: psPAX2 packaging plasmid: pLKO.1 shRNA expressing transfer plasmid with 17ul Lipofectamine 2000 in a 6-well plate well.  Media was changed 6 hours post transfection, and the viral containing media was collected and replaced at 48 and 72 hours post transfection.  Viral containing media was centrifuged at 600g for 5 min and the viral supernatant was collected and stored at 4°C prior to transduction.  Lentiviral transduction was performed on senescent BJ fibroblasts at ~50% confluency.   pLKO.1 stable knockdown lines were generated using a 48 ul : 1952 ul ratio of pLKO.1 viral supernatant with fresh culture media.  All transduction mixtures were given to cells and allowed to incubate overnight with polybrene added at a final concentration of 8ug/ml.  Knockdown cells were then maintained in 0.6ug/ml puromycin starting 4 days after transduction.  High Passage Growth Arrested Cells were transduced while still being maintained in rapamycin (10nM), and proceeded to the 2 week rapamycin maintenance/withdrawal phase of the experiment 14 days after the initial transduction.  Cells then underwent 14 days of additional 10nM rapamycin maintenance or withdrawal from rapamycin, both under continued 0.6ug/ml puromycin treatment.

value: High Passage Growth Arrested

value: Rapamycin Withdrawal


title: TSC421_BJ_HPGA_RW_shGFPO_Rep3

value: BJ Fibroblast


title: HEK293T, 1% Triton, Rep3, Supernatant, RNA-seq

value: embyonic kidney cells

value: 1% Triton


title: Subj.PNMB202_Day0


title: HEK293T, 1% Triton, Rep3, Pellet, RNA-seq

value: WT


title: MPA_UFRT_pospos_Rep3

description: Library name: KAR014_S29

value: MPA


title: Huh7 cell, iCLIP GPATCH4 rep2

city: shanghai

first: yiming

last: zhao

address: 320 Yue Yang Road

institute: Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences

data_processing:
the barcoded regions in the reads were filtered by FASTX-Toolkit. The demultiplexing and adapter trimming were performed by Flexbar. Only trimmed reads with a length at least 15 nt were kept for further analysis
The individual FASTQ files for each sample were mapped to the human reference genome (GRCh38) by STAR. The technical duplicates were removed using UMI-tools.
After deduplication, the crosslink events were transformed from the mapped reads by extracting the position upstream of the 5’ end of the reads with BEDtools .
Reproducibility of crosslink sites was determined by counting the percentage of crosslink sites that were present in two replicates.
The crosslink sites with significantly enriched crosslink events were identified by PureCLIP.
Assembly: GRCh38
Supplementary files format and content: bw file includes crosslink site for each sample

growth_protocol: Huh7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS)

extract_protocol:
Huh7 cells were irradiated once with 125 mJ/cm2at 254 nm. The iCLIP lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, 1× cOmpleteprotease inhibitors) was added to resuspend cell pellets. Cell lysates were sonicated in a Bioruptor (Diagenode), and then RNAs were partially fragmented with either high or low concentration of RNase A (Qiagen). The RNA-protein complexes were IP-ed with anti-FLAG antibody immobilized on protein G dynabeads (Invitrogen), and washed with High salt wash buffer (50 mM Tris-HCl pH 7.4, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, 0.5% Sodium Deoxycholate) and PNK buffer (20 mM Tris-HCl pH 7.4, 10 mM MgCl2, 0.2% Tween-20). 3’ End of RNAs were dephosphorylated and ligated to a L7-linker. RNAs were then radioactively labeled by T4 polynucleotide kinase (Thermo), and transferred from a NuPAGE4-12% Bis-Tris protein gel (Invitrogen) to a nitrocellulose membrane (Whatman). The RNA-protein complexes from a low concentration of RNase A treatment were recovered from the membrane
RNA reverse transcribed with RTclip2.0 primer. cDNA was purified and ligated at 5’ end with Lclip2.0 linker containing a certain barcode. For iCLIP cDNA library amplification, cDNA was first amplified with P5Solexa_s and P7Solexa_s, and PCR products were size selected with ProNex® chemistry. Products from the first amplification were then re-amplified with P5Solexa and P7Solexa and size selected.

value: HCC cell line

value: GPATCH4-FLAG stably expressed


title: Huh7 cell, iCLIP GPATCH4 rep3


title: Huh7 cell, iCLIP GPATCH4 rep1


title: MPA_UFRT_pospos_Rep2

description: Library name: KAR013_S28


title: HEK293T, 1% Triton, Rep3, Ribo-Seq


title: TSC402_BJ_HPGA_RM_shGFPN_Rep4

value: Rapamycin Maintained

value: shRNA - GFP - Control


title: TSC401_BJ_HPGA_RM_shGFPO_Rep3


title: TSC123_BJ_HPGA_RW_shNFIA_Rep1


title: Subj.PNMA101_Day0


title: HEK293T, 1% Triton, Rep2, Pellet, RNA-seq


title: Huh7 cell, iCLIP ctrl rep3

value: wild type


title: UFRT_D8_pospos_Rep4

description: Library name: KAR004_S19

value: Pseudopregnancy


title: MPA_UFRT_pospos_Rep1

description: Library name: KAR012_S27


title: HEK293T, 1% Triton, Rep2, Supernatant, RNA-seq


title: HEK293T, 1% Triton, Rep2, Ribo-Seq


title: UFRT_D8_pospos_Rep5

description: Library name: KAR005_S20


title: HEK293T, 1% Triton, Rep1, Pellet, RNA-seq


title: Subj.PNMB202_Day7


title: Subj.PNMA101_Day7


title: HEK293T, 1% Triton, Rep1, Supernatant, RNA-seq


title: UFRT_D8_pospos_Rep3

description: Library name: KAR003_S18


title: Huh7 cell, iCLIP ctrl rep1


title: TSC121_BJ_HPGA_RW_shGFP_Rep1


title: TSC130_BJ_HPGA_RW_shGFP-Rep2


title: UFRT_D8_pospos_Rep2

description: Library name: KAR002_S17


title: HEK293T, 1% Triton, Rep1, Ribo-Seq


title: A549-Flag-GFP, control

city: Shenyang

first: Man

last: Zhang

state: Liaoning

address: No. 66, Chongshan Zhonglu

institute: Liaoning University

data_processing:
The quality of the sequencing data was assessed with the Fast QC software (v.0.10.1). Double-ended clean reads were aligned with the reference genome using Hisat2 v.2.0.5
Gene expression was quantified with Counts v.1.5.0-p3 to calculate the number of reads mapped to each gene
Assembly: Homo Sapiens(GRCh37/hg19)
Supplementary files format and content: xlsx

source_name: Human lung adenocarcinoma

growth_protocol: Human lung adenocarcinoma A549 cells and human embryonic kidney 293T cells were obtained from the State Key Laboratory of Virology of the Wuhan University. A549 cells were cultured in F12K medium (Solabia, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA) and penicillin-streptomycin (Sigma, USA); 293T cells were cultured in high-glucose medium (HyClone, USA) supplemented with 10% FBS and penicillin-streptomycin. A549 cells were grown in 25-cm2 culture flasks to 80% confluence before collection.

extract_protocol:
Total RNA of each sample was extracted using TRIzol Reagent (Invitrogen)/RNeasy Mini Kit
Library construction and sequencing were performed on the Illumina HiSeq 2000 platform (Illumina, USA) at Golden Viz Biotech Ltd. to obtain paired-end reads 150 bp in length. Low-quality reads were removed according to the repository requirements.

value: Human lung adenocarcinoma

value: tumor cell


title: A549-Flag-GFP-NS1, OE H5N1 NS1


title: A549 cell, control


title: monocyte-derived Langerhans cells - BNP - 23D25

city: Debrecen

first: Attila

middle: Gábor

last: Szöllősi

address: Egyetem tér 1.

department: Department of Immunology

country: Hungary

institute: University of Debrecen

description: Library name: Sample 14

data_processing:
HISAT2 algorithm was used for alignment of raw sequencing reads to human reference genome version GRCh38.
Aligned data were normalized by using limma-voom algorithm, then differentially expressed genes were determined by moderated T-test. P value < 0.05 was considered to significant change.
Assembly: Assembly: GRCH38 human reference genome
Supplementary files format and content: Tab-delimited text file with gene IDs and read counts, with each sample in separate columns.

source_name: periphereal blood monocytes

growth_protocol: CD14+ monocytes were  cultured in RPMI 1640 medium containing 10% heat-inactivated FBS, 10% HEPES, 50 mM 2-mercaptoethanol, 1% penicillin. The cells were seeded in 12-well tissue-culture plates (1 000 000 per well) containing  media supplemented with GM-CSF (200 ng/mL), TGF-β1 (10 ng/mL), TNF-α (20 ng/mL), and IL-4 (20 ng/mL for the first 48 hrs) for 5 days at 37 °C to differentiate moLCs.

extract_protocol:
Trizol extraction of total RNA was performed according to the manufacturer's instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: BNP was  applied on day 0, day 2 and day 4  to relevant samples. Control cells were treated with appropriate vehicle on the same  days.

value: periphereal blood monocytes

value: BNP treated LC


title: HEK293T, 0.5% Triton, Rep3, Pellet, RNA-seq

value: 0.5% Triton


title: TSC120_BJ_HPGA_RM_shGFP-Rep2

value: High Passage Growth Arrested


title: Ercc1 d/- low fat - high carb #4

city: Utrecht

first: Wilbert

last: Vermeij

address: Heidelberglaan 25

country: Netherlands

institute: Princess Máxima Center

description:
Library name: 104750-001-019
019_GAACGGTT_ATTCCTCC

data_processing:
Analysis of raw data files was performed on our in house-generated data analysis pipeline. Removal of sequence adaptors from sequence reads was performed using Trimmomatic (version 0.39). Trimmed reads were aligned to mouse reference genome (annotation: gencode.vM20.annotation.gtf; genome: GRCm38.p6.genome.fa; http://gencodegenes.org/mouse/release_M20.html) using STAR (version 2.7.0f). Read counts for each gene were obtained using FeatureCounts (as part of SubRead version 1.6.4), filtered using filterByExpr with count set to 10, and followed by TMM normalization and quantification of log fold changes and false discovery rates using EdgeR (version 3.32.1).
Assembly: GRCm38.p6.genome.fa gencode.vM20.annotation.gtf
Supplementary files format and content: Tab-delimited count files produced by featureCounts

source_name: Liver

growth_protocol: Housing conditions Animals were maintained in a controlled environment (20–22°C, 12h light/dark cycle) and were housed in individual ventilated cages under specific pathogen‐free conditions. Animals were group housed  when possible. Since the Ercc1Δ/− mice are smaller, food was administered within the cages and water bottles with long nozzles were used from around two weeks of age. All animals were bred and maintained on AIN93G synthetic pellets (Research Diet Services B.V., Wijk bij Duurstede, Netherlands; gross energy content 4.9 kcal/g dry mass, digestible energy 3.97 kcal/g, using 2.3 g/kg choline chloride instead of choline bitartrate) until diet start. Macronutrient altered diets were provided AL starting at 8 weeks of age (see experimental set-up), continuing for the remainder of life. Animals were randomized across diets prior to the start of the interventions. Diets were processed into pellets and irradiated with 9 kGy (Isotron, Ede, the Netherlands). Food and water were offered ad libitum (AL) unless stated otherwise.

extract_protocol:
Snap-frozen liver tissues of 11-week-old Ercc1∆/− mice on diets with different macromolecular compositions were weighted to 30 mg and homogenized in QIAzol Lysis Reagent using a Qiagen tissuelyser LT. RNA was isolated using the Qiagen miRNeasy RNA isolation kit (QIAgen; 217004) following standard protocol with optional DNase (Qiagen, #79254) digestion step. RNA quality and quantity were assessed using the NanoDrop One (Thermo Fisher Scientific, ND-ONE-W) and Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA; G2939BA), respectively. Samples were sent for transcriptome sequencing (GenomeScan, Leiden, The Netherlands) on the Illumina NovaSeq 6000 platform.
Performed by GenomeScan using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina

treatment_protocol: See growth protocol.

tag: strain

value: F1 Hybrid C57BL6J/FVBN

value: Liver

value: Male

value: Ercc1 d/-

value: low fat - high carb #4


title: HEK293T, 0.5% Triton, Rep3, Supernatant, RNA-seq

value: embyonic kidney cells

value: WT


title: TSC111_BJ_HPGA_RM_shGFP_Rep1

value: BJ Fibroblast

value: Rapamycin Maintained


title: TSC122_BJ_HPGA_RW_shp65_Rep1

value: Rapamycin Withdrawal

value: shRNA - p65 - Construct 1


title: UFRT_D8_pospos_Rep1

description: Library name: KAR001_S16

value: Upper reproductive tract

value: Female

value: NK cell (Live lymphocytes, CD45.2+, DX5+, NK1.1+, Ly6G-, CD3-, CD19-)

value: Pseudopregnancy


title: TSB506_BJ_Senescent_shJMJD6-53

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:4 8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.    Long term shRNA experiments were done by transducing either pLKO.1 or GIPZ lentivirus into young (<PD30) BJ fibroblasts.  For both pLKO.1 and GIPZ constructs in young fibroblasts, cells were maintained under selection by 0.6ug/ml of puromyin and expanded for 2 weeks until they were frozen down.  Frozen stable lines were then thawed and grown continuously until reaching senescence, and the timing of their population doublings were recorded over time.

treatment_protocol: Lentivirus was produced through transfection of packaging plasmids into 293T (ATCC® CRL-3216) cells grown to ~80% confluency.  All transfections were done using Lipofectamine 2000 (Thermo Fisher 12566014), following the manufacturer’s protocol, with the ratios of plasmids and Lipofectamine 2000 reagent listed below.  GIPZ stable knockdown lines were generated using a 667ul : 1333ul ratio of GIPZ viral supernatant to fresh culture media.  pLKO.1 stable knockdown lines were generated using a 48 ul : 1952 ul ratio of pLKO.1 viral supernatant with fresh culture media.  All transduction mixtures were given to cells and allowed to incubate overnight with polybrene added at a final concentration of 8ug/ml.  Knockdown cells were then maintained in 0.6ug/ml puromycin starting 4 days after transduction, until they were harvested at the senescent stage.

value: Senescent

value: shRNA - JMJD6-construct 53


title: Ercc1 d/- low fat - high carb #3

description:
Library name: 104750-001-017
017_GTACACCT_CATGAGGA

value: F1 Hybrid C57BL6J/FVBN

value: low fat - high carb #3


title: Huh7 cell, iCLIP ctrl rep2

value: HCC cell line

value: wild type


title: monocyte-derived Langerhans cells - BNP - 23D24

description: Library name: Sample 13

value: periphereal blood monocytes

value: BNP treated LC


title: HEK293T, 0.5% Triton, Rep2, Pellet, RNA-seq

value: embyonic kidney cells

value: WT

value: 0.5% Triton


title: HEK293T, 0.5% Triton, Rep3, Ribo-Seq


title: Ercc1 d/- low fat - high carb #2

description:
Library name: 104750-001-010
010_TTACGTGC_CAAGGTCT

value: Liver

value: Male

value: Ercc1 d/-

value: low fat - high carb #2


title: TSB504_BJ_Senescent_shNS

value: BJ Fibroblast

value: shRNA - Non-silencing - Control


title: monocyte-derived Langerhans cells - BNP - 23D23

description: Library name: Sample 12


title: TSB541_BJ_PD64_Rapa

treatment_protocol: For long term rapamycin and EPZ-5676 DOT1L inhibitor treatments, young (<PD30) BJ fibroblasts were maintained in DMSO, 10nM rapamycin, 500nM EPZ-5676, or a combined 10nM rapamycin and 500nM EPZ-5676.  Cells’ population doubling were recorded, and cells were harvested for RNA-seq and ChIP-seq at young, intermediate, and senescent population doublings.

value: Senescent

value: Rapamycin


title: Ercc1 d/- low carbohydrate - high protein #4

description:
Library name: 104750-001-013
013_CAACTCCA_ACGGATTC

value: F1 Hybrid C57BL6J/FVBN

value: low carbohydrate - high protein #4


title: TSB540_BJ_PD64_EPZ500


title: HEK293T, 0.5% Triton, Rep2, Ribo-Seq


title: Ercc1 d/- low fat - high carb #1

description:
Library name: 104750-001-009
009_CGTATCTC_GTACCTTG

value: low fat - high carb #1


title: monocyte-derived Langerhans cells - BNP - 20D40

description: Library name: Sample 11


title: monocyte-derived Langerhans cells - BNP - 20D39

description: Library name: Sample 10


title: TSB539_BJ_Senescent_PD64_DMSO

value: DMSO


title: TSB542_BJ_PD64_E+R

value: Rapamycin + EPZ-5676


title: HEK293T, 0.5% Triton, Rep1, Pellet, RNA-seq


title: monocyte-derived Langerhans cells - CTRL - 23D25

description: Library name: Sample 7

value: periphereal blood monocytes

value: control moLC


title: HEK293T, 0.5% Triton, Rep1, Supernatant, RNA-seq

value: embyonic kidney cells

value: WT


title: monocyte-derived Langerhans cells - BNP - 20D36

description: Library name: Sample 9

value: BNP treated LC


title: TSB537_BJ_Intermediate_PD48.2_Rapa

value: Intermediate - Quiescent


title: Ercc1 d/- low carbohydrate - high protein #3

description:
Library name: 104750-001-006
006_GGCAAGTT_CTTCTGAG

value: Liver

value: Male

value: Ercc1 d/-

value: low carbohydrate - high protein #3


title: TSB538_BJ_Intermediate_PD48.2_E+R

value: BJ Fibroblast


title: Ercc1 d/- low carbohydrate - high protein #2

description:
Library name: 104750-001-002
002_CACAGACT_TGGTACAG

value: low carbohydrate - high protein #2


title: Ercc1 d/- low carbohydrate - high protein #1

description:
Library name: 104750-001-001
001_CGCAACTA_GAATCCGA

value: F1 Hybrid C57BL6J/FVBN

value: low carbohydrate - high protein #1


title: monocyte-derived Langerhans cells - CTRL - 23D24

description: Library name: Sample 6


title: Ercc1 d/- low protein - high carb #4

description:
Library name: 104750-001-012
012_AAGACACC_TCGGTTAC

value: low protein - high carb #4


title: HEK293T, 0.5% Triton, Rep1, Ribo-Seq

value: 0.5% Triton


title: TSB536_BJ_Intermediate_PD48.2_EPZ500


title: monocyte-derived Langerhans cells - CTRL - 23D23

description: Library name: Sample 5


title: HEK293T, 0.5% Triton, Rep2, Supernatant, RNA-seq


title: HEK293T, 0.1% Triton, Rep3, Supernatant, RNA-seq

value: 0.1% Triton


title: TSB534_BJ_Young_PD32_E+R

value: Young - Quiescent

value: Rapamycin + EPZ-5676


title: monocyte-derived Langerhans cells - CTRL - 20D40

description: Library name: Sample 4

value: periphereal blood monocytes

value: control moLC


title: TSB535_BJ_Intermediate_PD48.2_DMSO

value: DMSO


title: Ercc1 d/- low protein - high carb #2

description:
Library name: 104750-001-005
005_CTCGAACA_GGAATTGC

value: low protein - high carb #2


title: HEK293T, 0.1% Triton, Rep3, Ribo-Seq

value: embyonic kidney cells

value: WT


title: monocyte-derived Langerhans cells - CTRL - 20D39

description: Library name: Sample 3


title: Ercc1 d/- low protein - high carb #1

description:
Library name: 104750-001-003
003_TGGAAGCA_GTACTCTC

value: Liver

value: Male

value: Ercc1 d/-

value: low protein - high carb #1


title: TSB533_BJ_Young_PD32_Rapa

value: BJ Fibroblast

value: Rapamycin


title: Ercc1 d/- AIN93G control #4

description:
Library name: 104750-001-024
024_CTTCGGTT_CTCTGGTT

value: F1 Hybrid C57BL6J/FVBN

value: AIN93G control #4


title: monocyte-derived Langerhans cells - CTRL - 20D36

description: Library name: Sample 2


title: monocyte-derived Langerhans cells - BNP - 20D34

description: Library name: Sample 8

value: BNP treated LC


title: TSB532_BJ_Young_PD32_EPZ500

value: Young - Quiescent


title: HEK293T, 0.1% Triton, Rep2, Supernatant, RNA-seq

value: 0.1% Triton


title: monocyte-derived Langerhans cells - CTRL - 20D34

description: Library name: Sample 1

value: periphereal blood monocytes


title: Ercc1 d/- AIN93G control #2

description:
Library name: 104750-001-016
016_CTCGACTT_GGTCAGAT

value: AIN93G control #2


title: L10mut_bottom_R2

city: Kansas City

first: Hua

last: Li

state: MO

address: 1000 E 50th Street

institute: Stowers Institute for Medical Research

library_source: other

data_processing:
Analysis of NGS data sets e.g. sgRNA abundance was achieved using MAGeCK v0.5.9.2.
uL16 mutant stabilization is defined as the log2 fold change (LFC) of sgRNA abundance in the RFP-high vs. RFP-low sorted populations.
Assembly: hg38
Supplementary files format and content: tab-delimited text file that includes the raw count for each sgRNA in each sample (CRISPRi_FLOW_rawcountstable.txt) and a reference file for the library used (20200513_library_1_2_unbalanced.csv)

molecule: other

growth_protocol: K562 cells were grown in RPMI-1640 with 25 mM HEPES, 2.0 g/L NaHCO3, 0.3 g/L L-Glutamine supplemented with 10% FBS, 2mM glutamine, 100 units/mL penicillin and 100μg/mL streptomycin

extract_protocol:
Genomic DNA was extracted using NucleoSpin Blood kits from Macherey-Nagel, following the manufacturer's instructions.
Total genomic DNA (gDNA) concentration was measured, and the corresponding number of PCR reactions were assembled to use 10 µg gDNA per reaction (~25 reactions per sample). PCR reactions were prepared with NEBNext Ultra II Q5 MasterMix, gDNA, Forward (CAAGCAGAAGACGGCATACGAGATGCGGCCGGCTGTTTCCAGCTTAGCTCTTAAA) and Reverse (AATGATACGGCGACCACCGAGATCTACACtcgccttaCGCGTATCCCTTGGAGAACCACC) primers. Note that the underlined sequence is an Illumina barcode and varies for each sample. The following PCR conditions were used: 1. Initial denaturation for 30 seconds at 98ºC, 2. Denaturation at 98ºC for 10 seconds, 3. Annealing/extension at 65ºC for 75 seconds, 4. Repeat steps 2-3 twenty-one more times, 5. Final extension at 65ºC for 5 minutes. The PCR product was purified using SPRIselect Reagent (Beckman Coulter).
Ryn type: Paired-end 50 with R1: 19, I1: 8, R2: 19, I2: 8. Sequencing primers: Read 1 custom primer oJR437 (CGCGTATCCCTTGGAGAACCACCTTGTTGG); Index Read 1 custom primer oJR438 (CCAACAAGGTGGTTCTCCAAGGGATACGCG) ; Read 2 custom primer oJR439 (GCGGCCGGCTGTTTCCAGCTTAGCTCTTAAAC). Spike in 20% PhiX to maintain diversity

treatment_protocol: Cells transduced with sgRNAs were selected by treating them with 1 µg/ml puromycin for 3 days.

value: lymphoblast cells

value: uL16 mutant RFP BOTTOM (low RFP), replica 2

tag: time


title: Ercc1 d/- AIN93G control #3

description:
Library name: 104750-001-018
018_CCAAGGTT_GCTATCCT

value: AIN93G control #3


title: HEK293T, 0.1% Triton, Rep3, Pellet, RNA-seq

value: embyonic kidney cells

value: WT


title: TSB628_BJ_HPGA_RW_Rep2

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:4 8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.  Cells grown in rapamycin were introduced to and maintained in 10nM rapamycin after recovery from thawing.  For rapamycin withdrawal experiments, rapamycin maintained cells were grown to arrest, which typically occurred between PD75-85.  These cells are termed High Passage Growth Arrested.  At this point, the cells were divided into two plates that were either maintained or withdrawn from rapamycin for 2 weeks until they were collected.

value: High Passage Growth Arrested

value: Rapamycin Withdrawal


title: L10mut_bottom_R1

value: uL16 mutant RFP BOTTOM (low RFP), replica 1


title: HEK293T, 0.1% Triton, Rep2, Ribo-Seq


title: Ercc1 d/- AIN93G control #1

description:
Library name: 104750-001-015
015_CTTCACTG_GTCCTAAG

value: Liver

value: Male

value: Ercc1 d/-

value: AIN93G control #1


title: Ercc1 d/- low protein - high carb #3

description:
Library name: 104750-001-011
011_AGCTAAGC_GTAACGAC

value: F1 Hybrid C57BL6J/FVBN

value: low protein - high carb #3


title: TSB531_BJ_Young_PD32_DMSO

value: BJ Fibroblast

value: Young - Quiescent

value: DMSO


title: TSB627_BJ_HPGA_RM_Rep2

value: Rapamycin Maintained


title: TSB626_BJ_HPGA_RW_Rep1


title: Neurons isolated from primary motor cortex, baseline

city: New York

first: Vilas

last: Menon

state: NY

department: Neurology

institute: Columbia University Medical Center

description:
Library name: RM13
10X Genomics

library_source: transcriptomic single cell

data_processing:
Read alignment and quantification using Cellranger version 3.1.0
Assembly: GRCm38 (mm10)
Supplementary files format and content: raw h5 counts tables for each sample

source_name: brain; primary motor cortex

extract_protocol:
After CaMPARI labeling, mice were returned to their home cage for 15 minutes and then euthanatized by transcardial perfusion. Subsequent steps were adapted from previous studies describing a protocol optimized for preserving transcriptional state57. Perfusions were performed with with ice-cold choline solution: 2.1 g/l NaHCO3, 2.16 g/l glucose, 0.172 g/l, NaH2PO4 * H2O, 7.5mM MgCl2•6H2O, 2.5mM KCl, 10mM HEPES, 15.36 g/l choline chloride, 2.3 g/l ascorbic acid, and 0.34 g/l pyruvic acid. Thick sections were cut using a matrix, and M1 was quickly dissected. The dissected M1 tissue was further triturated with spring scissors. The tissue was then digested with the Papain dissociation system (Worthington) according to the manufacturer’s instructions. However, the EBSS solution was replaced with the following: HBSS (Life Technologies), 10mM HEPES (Sigma), 172mg/l kynurenic acid (Sigma), 0.86 g/l MgCl2•6H2O (Sigma), 6.3 g/l D-glucose (Sigma). Incubation with papain was conducted using a dialysis membrane (Fisher Scientific, Slide-a-Lyzer MINI Dialysis Device,10k molecular weight cut-off) suspended in a beaker of dissociation solution allowing for constant oxygenation (95% O2 and 5% CO2) of the solution without disrupting enzyme integrity within the dialysis membrane. After enzymatic dissociation, the tissue was broken into a single-cell suspension by gently pipetting using wide-bore pipette tips. The sample was cleaned using the Debris Removal Solution (Miltenyi Biotec) as described by the manufacturer. Cells were suspended in dissociation solution containing 0.04% BSA, Vybrant DyeCycle Ruby for exclusion debris, and RNAase inhibitors. Cell sorting was performed using a FACSAria Cell Sorter (Becton Dickinson) with a 130 um nozzle at 12 PSI using the following laser lines and filters: CaMPARI Green, 488 nm, 530/30 bandpass; CaMPARI Red, 561 nm, 610/20 bandpass; DyeCycle Ruby, 637 nm, 670/30 bandpass. Gates were set using a sample not expressing CaMPARI and a sample not exposed to photoconversion light. Any cells with detectable red fluorescence were collected.
10x Genomics Chromium 3' end v3

value: brain; primary motor cortex

value: neurons

value: male

tag: age

value: 12-16 weeks


title: L10mut_top_R1

value: lymphoblast cells

value: uL16 mutant RFP TOP (high RFP), replica 1


title: HEK293T, 0.1% Triton, Rep2, Pellet, RNA-seq

value: embyonic kidney cells

value: WT

value: 0.1% Triton


title: Mutant iN replicate 2

city: Piscataway

first: Ronald

middle: P.

last: Hart

state: NJ

address: 604 Allison Rd Rm B430

department: Cell Biology & Neuroscience

institute: Rutgers University

data_processing:
Raw fastq sequencing files were aligned with human reference genome (hg38) using HISAT2
Raw counts of expressed genes were extracted using featureCounts from the Rsubread package.
Counts were normalized, scaled, and fit to a model in DESeq2
Processed data table extracted with fpm() function
Differential gene expression (mutant vs. wild-type) was extracted from the DESeq2 object
Assembly: hg38
Supplementary files format and content: samples x genes, fpm

growth_protocol: Induced neurons were cultured for  5–7 days in B27- and doxycycline-containing neurobasal medium before harvesting.

extract_protocol:
Total cellular RNA was harvested with TRIzol Reagent (Thermo Fisher Scientific) and treated with RNase-free DNase (Promega) following manufacturers’ protocols.
Illumina TruSeq 3' RNA Library kits were used.

value: Induced neuron

value: DCPS-null mutant


title: scRNAseq CCl4

city: ROCHESTER

first: William

last: Sherman

state: MN

address: 200 1st St SW

department: Quantitative Health Sciences

institute: Mayo Clinic

description:
Library name: out_CCl4
10X Genomics (cDNA)

data_processing:
scRNAseq fastq files were processed via CellRanger 4.0.0 and mapped to mm10 genome.
Assembly: mm10
Supplementary files format and content: .h5 filtered feature matrix

growth_protocol: Starting at 8 weeks of age, C57B6 mice were administered 1 mL/g body weight carbon tetrachloride (CCl4) or olive oil  twice weekly  for 6 weeks to induce fibrosis before sacrifice.

extract_protocol:
Mice non-parenchymal cells were isolated using previously published protocols. Primary mice hepatocytes were isolated using a modification of the two-step collagenase perfusion method. Cell viability was >95% as measured by Trypan Blue staining.
For scRNAseq:Samples were then prepared as outlined by the 10x Genomics Single Cell 3v2 Reagent Kit user guide. Briefly, the samples were washed twice in PBS (Life Technologies) + 0.04% BSA (Sigma) and re-suspended in PBS + 0.04% BSA. Following counting, the appropriate volume for each sample was calculated for a target capture of 12000 cells. Samples were loaded onto the 10x Genomics single-cell-A chip. After droplet generation, samples were transferred onto a pre-chilled 96-well plate (Eppendorf), heat-sealed and reverse transcription was performed using a Veriti 96-well thermal cycler (Thermo Fisher). After the reverse transcription, cDNA was recovered using Recovery Agent provided by 10x followed by a Silane DynaBead clean-up (Thermo Fisher) as outlined in the user guide. Purified cDNA was amplified for 12 cycles before being cleaned up using SPRIselect beads (Beckman). Samples were diluted 4:1 (elution buffer (Qiagen): cDNA) and run on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. cDNA libraries were prepared as outlined by the Single Cell Reagent Kits v2 user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration (as recommended by 10X Genomics).
The molarity of each library was calculated based on library size as measured using a bioanalyzer (Agilent Technologies) and qPCR amplification data (Kappa/Roche). Samples were pooled and normalized to 10 nM, then diluted to 2 nM using elution buffer (Qiagen) with 0.1% Tween20 (Sigma). Each 2 nM pool was denatured using 0.1 N NaOH at equal volumes for 5 min at room temperature. Library pools were further diluted to 20 pM using HT-1 (Illumina) before being diluted to a final loading concentration of 14 pM. 150 μl from the 14 pM pool was loaded into each well of an 8-well strip tube and loaded onto a cBot (Illumina) for cluster generation. Samples were sequenced on a HiSeq 2500 with the following run parameters: Read 1—26 cycles, read 2—98 cycles, index 1—8 cycles.

value: Liver

value: Mixed liver single cell suspension


title: L10mut_top_R2

value: uL16 mutant RFP TOP (high RFP), replica 2


title: TSB625_BJ_HPGA_RM_Rep1

value: BJ Fibroblast

value: High Passage Growth Arrested


title: Neurons isolated from primary motor cortex, late learning (biol rep 5)

description:
Library name: RM14
10X Genomics


title: HEK293T, 0.1% Triton, Rep1, Pellet, RNA-seq


title: scRNAseq Healthy

description:
Library name: out_Healthy
10X Genomics (cDNA)

value: Control


title: Mutant iN replicate 3


title: Neurons isolated from primary motor cortex, control (biol rep 4)

description:
Library name: RM11
10X Genomics

value: brain; primary motor cortex


title: HEK293T, 0.1% Triton, Rep1, Ribo-Seq


title: HEK293T, 0.1% Triton, Rep1, Supernatant, RNA-seq


title: Mutant iN replicate 1


title: TSB623_BJ_Sen_dox-

growth_protocol: BJ fibroblasts (ATCC® CRL-2522) were cultured at 37 °C in 20% O2 and 5% CO2 in 4.5 g/L DMEM (Gibco 10566-016) in 10% FBS.  Cell lines were initially plated at PD26, grown to confluence, and then split 1:8.  Population doubling was recorded and incremented by 3 upon each 1:8 split.  BJ fibroblasts were considered to be “young” when collected below population doubling (PD) 35, intermediate fibroblasts were harvested between PD 35-50, and fibroblasts were considered to be senescent when cells failed to divide after at least one week of passaging, which typically occurred after at least PD64 and was initially confirmed using beta galactosidase staining.  To mitigate the effects of cell cycle upon our results, non-senescent, still dividing fibroblasts were grown to confluence prior to harvesting for any experiments, in order to ensure contact inhibited quiescence and similar growth arrest among all populations of cells.  Tet-inducible overexpression experiments (empty vector, in this case) were done by transducing a combination of pRetroX and TetOn retrovirus into young (<PD30) BJ fibroblasts.  3 days after transduction, cells were maintained under combined selection by 0.6 ug/ml puromycin (Gibco A1113803) and 0.3mg/ml G418 (GeminiBio 400-113) and expanded for 2 weeks until they were frozen down.  Frozen stable lines were then thawed and grown in the continued presence or absence of doxycycline (20nM).

treatment_protocol: Frozen stable lines were then thawed and grown either under either continuous treatment of 20nM doxycycline or no doxycycline treatment.

value: Senescent

value: Doxycycline Minus


title: TSB624_BJ_Sen_dox+

value: Doxycycline Plus


title: Exp2_Prkar1a KD + Gcgr mAb_rep10

city: Tarrytown

first: Kehan

last: Bao

address: 777 Old Saw Mill River Rd

institute: Regeneron Pharmaceuticals

data_processing:
Raw sequence data (BCL files) were converted to FASTQ format via Illumina bcl2fastq v2.17. Reads were decoded based on their barcodes, and read quality was evaluated with FastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Reads were mapped to the mouse genome (NCBI GRCm38) using ArrayStudio® software (OmicSoft®, Cary, NC) allowing two mismatches. Reads mapped to the exons of a gene were summed at the gene level.
Assembly: GRCm38/mm10
Supplementary files format and content: csv files of raw counts for each sample

source_name: liver

extract_protocol:
Total RNA was purified from liver tissue using MagMAX-96 for the Microarrays Total RNA Isolation kit (Ambion by Life Technologies), according to manufacturer specifications. Genomic DNA was removed using MagMAX Turbo DNase buffer and TURBO DNase from the MagMAX kit listed above (Ambion by Life Technologies). mRNA was purified from total RNA using the Dynabeads® mRNA Purification kit (Invitrogen).
Strand-specific RNA sequencing (RNA-seq) libraries were prepared using the KAPA mRNA-Seq Library Preparation Kit (Kapa Biosystems). Twelve-cycle PCR was performed to amplify libraries.

value: liver

value: Prkar1a KD + Gcgr mAb0


title: TSB622_BJ_Young_dox+

city: La Jolla

first: Tom

last: Suter

institute: UCSD

data_processing:
Data was aligned to hg18 using STAR-aligner, with parameters adjusted to account for spliced alignment.
Aligned reads were processed for downstream analysis using the HOMER (Heinz et al., 2010) “makeTagDirectory” program, filtering for tags that were uniquely mapped and only selecting at most one sequencing read per position per length of read.
bigWig files were generated using the makeBigWig.pl function in HOMER, using the -rnaseq style parameter.
Genome_build: hg18
Supplementary_files_format_and_content: .bigWig : display of ChIP-seq data on genome browser as a graph

extract_protocol:
Total cellular RNA was harvested from cells using the RNeasy Mini Kit (QIAGEN-74104), making use of the optional DNase treatment step.
Samples were then prepared for sequencing with the Illumina HiSeq 2500 system using the TruSeq Stranded Total RNA LT Sample Prep Kit (RS-122-2201/RS-122-2202) for ribosomal RNA depleted libraries.  Samples were run on the Illumina HiSeq 2500 as single-read reactions of 50 cycles.

value: Young - Quiescent


title: Neurons isolated from primary motor cortex, early learning (biol rep 4)

description:
Library name: RM10
10X Genomics

value: neurons

value: WT

value: male

value: 12-16 weeks


title: Exp2_Control AAV + Control mAb_rep10

value: Control AAV + Control mAb0


title: Wild-type iN replicate 3

value: Induced neuron

value: wild-type


title: Neurons isolated from primary motor cortex, late learning (biol rep 4)

description:
Library name: RM12
10X Genomics


title: TSB621_BJ_Young_dox-

value: BJ Fibroblast


title: Exp2_Prkar1a KD + Control mAb_rep10

value: Prkar1a KD + Control mAb0


title: Neurons isolated from primary motor cortex, early learning (biol rep 2)

description:
Library name: RM08
10X Genomics


title: Wild-type iN replicate 2


title: Wild-type iN replicate 1


title: TSC354_BJ_ATAC_shNFIX-64

data_processing:
Data was aligned to hg18 using STAR-aligner with parameters adjusted avoid spliced alignments.
Aligned reads were processed for downstream analysis using the HOMER (Heinz et al., 2010) “makeTagDirectory” program, filtering for tags that were uniquely mapped and only selecting at most one sequencing read per position per length of read.
bigWig files were generated using the makeBigWig.pl function in HOMER
Genome_build: hg18
Supplementary_files_format_and_content: .bigWig : display of ATAC-seq data on genome browser as a graph

extract_protocol:
~50,000 BJ fibroblasts (senescent or quiescent via contact inhibition) were collected via trypsinization, washed in PBS on ice, resuspended in 200uL ice cold ATAC-seq lysis buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 3mM MgCl2, 0.1% Igepal CA-630) by being gently pipetted up and down 10 times, and immediately centrifuged for at 1000g for 10 minutes at 4°C.  The tagmentation reaction was performed using the Nextera DNA Library Prep Kit (Illumina-FC-121-1030), with the pellet being resuspended in 50uL 1x TD buffer and with 2.5uL Tn5 Transposase.  This reaction was incubated at 37°C for 30 minutes, after which DNA was purified using MinElute Reaction Cleanup Kit (Qiagen-28204).
Tagmented DNA was prepared for Illumina sequencing using the KAPA Real-Time Library amplification kit (KAPA/Roche-KK2701), as per manufacturer’s instructions, using Illumina Nextera primers.  Individual samples’ reactions were removed upon their qPCR intensity reaching that of Standard 2, which was typically after around 10 cycles of amplification.  Once all samples were sufficiently amplified and removed, the samples were size selected for 150-800bp fragments using AmPureXP beads (Beckman-A63880).  Samples were then sequenced using Illumina’s Nextera sequencing primer on the HiSeq 2500 using 100 cycle single read runs.

tag: shRNA target

value: shRNA - NFIX


title: Exp2_Control AAV + Gcgr mAb_rep10

value: Control AAV + Gcgr mAb0


title: Neurons isolated from primary motor cortex, control (biol rep 3)

description:
Library name: RM07
10X Genomics

value: brain; primary motor cortex