title: Differentiating, normal glucose, DDX21 epitope, donor 1, input control


title: RRBS_TSC1_WT_single_cell_sorted_cl11

status: Public on Jan 02 2023

type: SRA

city: Berlin

first: Sara

last: Hetzel

address: Ihnestraße 63

department: Genome Regulation

country: Germany

institute: Max Planck Institute for Molecular Genetics

description: Bisulfite-converted DNA

library_source: genomic

data_processing:
Raw reads were subjected to adapter and quality trimming using cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25; Illumina TruSeq adapter), followed by NuGEN diversity adapter trimming (https://github.com/nugentechnologies/NuMetRRBS).
The trimmed reads were aligned to the mouse genome (mm10) using BSMAP (version 2.90; parameters: -v 0.1 -s 12 -q 20 -w 100 -S 1 -u -R -D C-CGG).
Aligned reads were deduplicated based on unique molecular identifiers (UMIs) using NuDup (https://github.com/nugentechnologies/nudup; parameters: --start 6 --length 6). Methylation rates were called using mcall from the MOABS package (version 1.3.2; default parameters).
All analyses were restricted to autosomes and only CpGs covered by at least 10 and at maximum 150 reads were considered for downstream analyses.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig file containing methylation rates for CpGs covered by at least 10 and at most 150 reads: <chr> <start> <end> <methylation rate>

molecule: genomic DNA

organism: Mus musculus

source_name: Trophoblast stem cells

growth_protocol: TSCs (TSC1 line, clone 11) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149).

extract_protocol:
The DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermo, #K182002) according to manufacturer’s instructions.
Concentration of genomic DNA (gDNA) was quantified using a Qubit 3.0 Fluorometer. RRBS was performed on 100 ng gDNA of each sample using the NuGen Ovation® RRBS Methyl-Seq System (Tecan, #0353) following the manufacturer’s recommendations with the following modifications (6). After the final repair step, the bisulfite conversion of DNA was conducted using the Qiagen EpiTect Fast Bisulfite Conversion kit (Qiagen, #59824) following the manufacturer’s recommendations, eluting the bisulfite converted DNA in 23 µl EB. Libraries were amplified with 12 cycles of PCR. Amplified library purification with Agencourt RNAclean XP beads (Beckman Coulter, #A63987) was performed twice (1X). The purified libraries were quality-assessed on an Agilent 4150 Tape-Station HS D1000 ScreenTape and sequenced for 100-bp single-end reads on the NovaSeq6000 (Illumina).

treatment_protocol: TSCs (TSC1 line, clone 11) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.

tag: condition

value: WT


title: Differentiating, normal glucose, DDX21 epitope, donor 1, ChIP-seq

city: Stanford

first: Douglas

last: Porter

state: CA

address: 269 Campus Drive

department: Dermatology

country: USA

institute: Stanford

data_processing:
Cutadapt (MARTIN, 2011) (v. 1.8.1) was used to remove NEBNext/TruSeq adapters (AGA TCG GAA GAG CAC ACG TCT GAA CTC CAG TCA and AGA TCG GAA GAG CGT CGT GTA GGG AAA GAG TGT).
Bowtie2 (Langmead and Salzberg, 2012) (v. 2.4.2) was used to map clipped reads to the hg38 genome (pre-built index grch38_1kgmaj), using the parameter --local. We then filtered out reads that were unmapped, duplicate, or had less than 10 mapping quality.
MACS2 (Gaspar, 2018; Zhang et al., 2008) (v. 2.2.71) was run on the filtered bam files, controlling ChIP samples to input samples, using the default parameters plus -f BAMPE --bdg. Peak regions across all samples were merged using bedtools (Quinlan and Hall, 2010) merge. Reads in peak regions were determined using bedtools multicov, passing the bam files and the merged peak file as parameters.
Genome_build: GRCh38
Supplementary_files_format_and_content: MACS2 called peak regions were subset to TSS peaks using the TxDb.Hsapiens.UCSC.hg38.knownGene package in R. Reads in each region are reported for each sample.

organism: Homo sapiens

source_name: Primary human foreskin keratinocytes

growth_protocol: Primary human keratinocytes were isolated from fresh surgically discarded newborn foreskin, and progenitor cells cultured in complete Keratinocyte-SFM (Life Technologies, #17005-142) and Medium 154 (Life Technologies #M-154-500). Cells were induced to differentiate by the addition of 1.2 mM calcium for 3 days at full confluence. Cells with low glucose and the corresponding normal glucose condition were maintained in glucose-free DMEM (Thermo Fishier, #11966025) supplied with SFM supplement (rEGF: Gibco, #10450-013; BPE: 13028-014), 1X Sodium Pyruvate (Gibco, 11360070) and a final concentration of 0.5 g/L (“low” glucose, Gibco, A2494001) or 4.5 g/L glucose (“normal” glucose) with adding 1.2 mM calcium during differentiation for 3 days. Biological replicates were performed in all cases using primary cells from at least 2 independent, unrelated donors.

extract_protocol:
Cells were lysed with 500 µL swelling buffer (100 mM Tris-HCl pH 7.5, 10 mM KOAc, 15 mM MgOAc, 1% NP-40 and 1X protease inhibitor, EDTA-free) on ice for 10 minutes and transfered into a pre-chilled 2 ml Dounce homogenizer (Type B) to break cells open using 10 strokes of a tight pestle. After centrifugation for 5 minutes at 4000 rpm, 4℃, nuclei were lysed with 10X the amount of nuclei lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA and 1X protease inhibitor, EDTA-free) on ice for 10 minutes, transfered to Polystyrene tubes and sonicated to shear the chromatin to 150-250bp (Bioruptor UCD-200).
40-100 µg sheared chromatin was input to either DDX21- or HA-immunopurification. Sheared chromatin was diluted 5-8-fold in ChIP dilution buffer (16.7 mM Tris-HCl, pH = 7.5, 167 mM NaCl, 1.2 mM EDTA, 1.1% TritonX-100, 0.01% SDS). 1 µg DDX21 antibody (Novus Biologicals, NBP1-83310) was added for each 20-µg chromatin and incubated overnight at 4℃. 60 µL protein G dynabeads (Invitrogen, 10004D) was used for each 50-µg sheared chromatin, and incubated for 2 hours at 4℃ followed by washing with buffer A (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% TritonX-100, 0.1% SDS, 0.1% NaDOC), buffer B (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% TritonX-100, 0.1% SDS, 0.1% NaDOC), buffer C (50 mM Tris-HCl pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% NaDOC) and finally buffer D (10 mM Tris-HCl, pH 8.0, 1 mM EDTA), each wash performed twice. Beads were then incubated at 65℃ with elution buffer (10 mM Tris-HCl pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.5% SDS) for 15 minutes. Eluted DNA was incubated at 67℃ overnight to reverse crosslinks, followed by adding 1 µL 10 mg/mL RNaseA (Qiagen, 1032724) and incubating at 37℃ for 30 minutes to remove RNA. The resulting DNA was further purified using a PCR purification kit (Qiagen, 28106) and quantified by Qubit High Sensitivity dsDNA kit (Invitrogen, Q32851). One µg of input DNA and ChIP DNA were input to ChIP library preparation using the NEBNext Ultra II DNA library prep kit (NEB, E7103S), following the manufacturer’s protocol.

treatment_protocol: Ten million cells were harvested and crosslinked by 1% formaldehyde in PBS at room temperature and quenched by 0.125 M Glycine for each ChIP experiment.

tag: treatment

value: Normal glucose

tag: donor

tag: growth


title: ifos1_D

city: Gainesville

first: Christopher

last: Martyniuk

state: FL

address: 8891 SW 79th Avenue

institute: University of Florida

library_source: transcriptomic

data_processing:
Raw data (raw reads) of FASTQ format were firstly processed through fastp. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. At the same time, Q20, Q30 and GC content of the clean data were calculated. All the downstream analyses were based on the clean data with high quality.
Reference genome and gene model annotation files were downloaded from genome website browser (NCBI/UCSC/Ensembl) directly.
Paired-end clean reads were mapped to the reference genome using HISAT2 software.
Featurecounts was used to count the read numbers mapped of each gene, including known and novel genes. RPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.
DESeq2 was used for differential expression analysis between the control and treatments.
Assembly: ensembl_danio_rerio_grcz11_gca_
Supplementary files format and content: text file containing read count, fpkm, log2 fold change, p-value

molecule: total RNA

organism: Danio rerio

source_name: larval zebrafish

extract_protocol:
Total RNA for each sample was isolated by using TRIzol. RNA concentration was determined using the Qubit® 2.0 Fluorometer (ThermoFisher/Invitrogen, Grand Island, NY), and RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). RNA samples with a measured 28S/18S > 1 and RNA integrity number (RIN) ≥ 7 were used for RNAseq library construction.
Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library
Total RNA was used for mRNA isolation using the NEBNext Ploy(A) mRNA Magnetic Isolation module (New England Biolabs, catalog # E7490). RNA library construction was then performed with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, catalog #E7760) according to the manufacturer's user guide. Individually prepared libraries were pooled by equimolar and sequenced by NovoSeq6000 (Illumina Inc., CA, U.S.A). Sequencng was performed by Novogene.

tag: tissue

value: larvae

value: 1 ug/L ifosfamide


title: ifos1_C


title: RRBS_TSC1_WT_single_cell_sorted_cl10

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 10) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149).

treatment_protocol: TSCs (TSC1 line, clone 10) were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.


title: SN2285_GFP_plus

city: Cambridge

first: A

last: Bhutkar

state: MA

address: 77 Massachusetts Avenue

institute: MIT

data_processing:
Illumina pipeline version 1.3+ used for basecalling and sequence read generation
Reads were trimmed to remove Illumina TruSeq adapter sequences using cutadapt (v1.16) and mapped to the mouse genome (UCSC annotation, genome build mm9) using STAR aligner v2.5.3a. Per gene read counts were quantified using the featureCounts utility in the R Subread package.
Assembly: mm9
Supplementary files format and content: Microsoft Excel file includes raw read counts (from featureCounts) per gene for each sample

molecule: polyA RNA

source_name: lung cells

growth_protocol: FNHCAS media in 90% matrigel drops (3D), maintained in a humidified 37ºC / 5% CO2 incubator at ambient O2 pressure

extract_protocol:
TRIzol Plus RNA Purification Kit (Invitrogen, 12183555)
RNAseq libraries were prepared from 300ng of total RNA using the Kapa Hyperprep kit (Roche) with 14 cycles of PCR

value: lung cells

tag: cell line

tag: cell type

value: Alveolar organoid

tag: genotype

value: SN2285_GFP+, eGFP+ lung cells from Sftpc-eGFP transgenic mice

value: FACS sorted for eGFP+


title: Fixed_D3_1589_Protein

city: Nijmegen

first: Jessie

middle: van

last: Buggenum

address: Heyendaalseweg 135

country: Netherlands

institute: Radboud University

data_processing:
Library strategy: CITE-seq
Bcl2fasq software (illumina) was used for data-demultiplexing.
The Celseq2 pipeline or CITE-seq-count tool were used to create count tables of mRNA or protein libraries respectively. Standard settings were used with some minor modifications. The Celseq2 pipeline was run using settings [min_bc_quality = 10, cut_length = 50]. Additionally, the read names for read 1 and read 2 were swapped to allow compatibility of our barcode design with the pipeline. The CITE-seq-count settings were kept at default with exeption of [ max_error of Ab barcode = 1, umi_collpasing_dist = 1].
Genome_build: GRCh38
Supplementary_files_format_and_content: For the mRNA modality and protein modality count tables were generated called CITEseq_####.txt and COUNTS_####.tab. In rows one can find features (transcript or protein respectively) and in each column is one cell

molecule: protein

source_name: Differentiated primary isolated B-cell

growth_protocol: B-cells were pre-enriched using the RossetteSep Human B-cell Enrichment cocktail according manufacturer’s instructions (StemCell, USA). After Ficoll density gradient total B-cells were further isolated using the B cell isolation Kit II according manufacturer’s instructions (Miltenyi Biotec, Germany).

extract_protocol:
Day 11 cells were harvested, counted and split in two tubes, one to fixed/permeabilize and one for live cells analysis. Fixed/permeabilized cells were immediately fixed by adding 2x concentrated fixative to the cell suspension (5 mM dithiobis (succinimidyl propionate) (DSP) (Thermo Fisher Scientific, USA) and 5 mM succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Thermo Fisher Scientific, USA) in PBS (Thermo Fisher Scientific, USA). Cells were incubated at room temperature for 45 minutes while gently shaking. Fixed cells were quenched and permeabilized with 100 mM Tris-HCl pH 7.5, 150 mM NaCl and 0.1% Triton X100 (Thermo Fisher Scientific, USA) for 10 minutes at room temperature. After permeabilization, cells were washed once and blocked for 45 minutes in 0.5X protein-free blocking buffer (Thermo Fisher Scientific, USA) with 0.2 mg/ml dextran sulfate (Sigma Aldrich, USA) and 0.5 U/ml RNasin plus (Promega, USA) in PBS (Thermo Fisher Scientific, USA). Cells were stained in a blocking buffer containing DNA-tagged antibodies (Supplementary Table 2) and PE-Cy7 conjugated CD38 Ab (Supplementary Table 1) for one hour at room temperature. Following staining, cells were washed twice with the blocking buffer and re-suspended in PBS containing 5 mg/ml BSA (Thermo Fisher Scientific, USA) and 0.5 U/ml RNsing plus (Promega, USA) and 0.1μg/ml DAPI (Biolegend, USA). Non-fixed cells were processed immediately and were washed twice in ice cold blocking buffer. Cells were stained in ice cold blocking buffer containing DNA-tagged Abs (Supplementary Table 3) and PE-Cy7 conjugated CD38 Ab (Supplementary Table 1). Cells were incubated on ice in the dark for 20min. Cells were washed three times in ice cold blocking buffer and re-suspended in blocking buffer containing 0.1μg/ml DAPI (Biolegend, USA). Cells were sorted single-cell in 384-wells PCR plates (Biorad, USA) using the FACS Melody (BD Biosciences, USA). Cells were sorted in 100nl water containing 7.5 ng/μl unique Cellseq-2 primers Supplementary Table 4) and 5 μl miniral oil (Sigma Aldrich, USA). Plates were stored at -80 °C until further use.
Plates were thawed on ice before further processing. Reverse crosslinking was performed by dispensing 50 nl of a 3X reverse-crosslinking buffer [6 mM dNTP, 150 mM Tris pH 8, 90 mM DTT, 0.1% Triton X100, 6 U/μl RNAsin Plus (Promega)] into the wells and incubating 45 minutes at 25 °C. Hereafter, the reactions were incubated 5 minutes at 65 °C and cooled to 4 °C. Next, reverse transcription was performed in a total reaction volume of 250 nl using the Maxima H minus Reverse transcriptase (Thermo Scientific). The samples were heated to 85 °C for 5 minutes and the slowly cooled down by decreasing the temperature 5 degrees every 30 seconds until reaching 10 °C. Next, the samples were pooled and purified. During purification using 0.6x AMPure XP machnetic beads (Beckman Coulter, USA, the smaller antibody barcodes were separated from the large mRNAs by size selection. After the larger mRNAs bound to the beads the supernatant containing the Ab barcodes was taken and prepared separately. For the mRNA part, second strand synthesis and in vitro transcription were performed as described. Reverse transcription of the amplified RNA was performed with Maxima H minus Reverse Transcriptase (Thermo Scientific) using a combination of a random octamer primer. Library preparation PCR was performed in two steps. First a library pre-amplification with short primers was performed using the Herculase II enzyme (Agilent) to minimize amplification bias. Next, adapter extension was performed using a PCR reaction with Herculase II (Agilent) and the following oligonucleotides , Reverse indexing oligonucleotide.  Protein library preparation After size separation of the protein and mRNA libraries, the protein library was purified with 1.5x AMPure XP magnetic beads and eluted in 10 µL of nuclease-free water. Subsequently, the sample was PCR amplified after prior determination of the number of cycles needed by qPCR: 0.5 µL of purified material was mixed with 6.5 µL nuclease-free water, 10 µL of 2x Kapa HiFi HotStart PCR mix (Roche, Switzerland), 1 µL of 20X EvaGreen Dye (Thermo Fisher Scientific, USA), and 2 µL of REV unique indexing primer and the Nextera primer, primer mix (5 µM each) (Biolegio, The Netherlands). Then the PCR reaction was started by adding: 0.5 µL of nuclease-free water, 12.5 µL of 2X Kapa HiFi HotStart PCR mix (Roche, Switzerland), and 2.5 µL of REV unique indexing primer and the Nextera primer mix (5 µM each) (Biolegio, The Netherlands) to the 9.5 µL of the sample. The PCR thermal cycling was as follows: 2 minutes at 95 °C, followed by x cycles of (x = number previously determined by qPCR): 30 seconds at 95 °C, 30 seconds at 64 °C and 40 seconds at 72 °C, and terminated with 5 minute-incubation at 72 °C. PCR amplified product was purified by addition of 1.2X AMPure XP magnetic beads, eluted in 10 µl water and its quality was verified by ds-DNA concentration measurement with Qubit ds-DNA High Sensitivity assay (Thermo Fisher Scientific, USA) and Bioanalyzer (Agilent 2100, USA) characterization. If the quality of the samples was high enough, the protein libraries were sequenced together with the mRNA libraries on with NextSeq500 (targeting 50 million reads per sample).

treatment_protocol: All B cell cultures were performed in Iscove's Modified Dulbecco's Medium (IMDM) (Thermo Fisher Scientific, USA) supplemented with 10% FBS (Thermo Fisher Scientific, USA) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific, USA). 2.5*10E6 purified B-cells were seeded in 500 μl medium in 14 ml round bottom tubes (VWR, the Netherlands). 500 μl medium was added containing 2x concentrated CpG ODN2006, Class B at 2 μg/ml (Invivogen, USA). Final concentration of CpG is 1 μg/ml in 1 ml volume. Cells were incubated for 16 hours at 37°C/5 % CO2. Cells were washed once by adding 4 ml of medium and centrifuging cells at 200 g for 5 min. Supernatant was removed completely and 5 ml of new medium was added containing 500 ng/ml rhCD40L (Miltenyi Biotec, Germany), 20 U/ml rhIL-2 (Sigma Aldrich, USA), 50 ng/ml rhIL-10 (R&D Systems, USA), 10 ng/ml rhIL-15 (R&D Systems, USA), 100 ng/ml rhIL-21 (Miltenyi Biotec, Germany). Cells were incubated for 3 days at 37°C/5 % CO2. After incubation, supernatant was removed completely and 5 ml of new medium was added containing 20 U/ml rhIL-2 (Sigma Aldrich, USA), 50 ng/ml rhIL-10 (R&D Systems, USA), 10 ng/ml rhIL-15 (R&D Systems, USA), 100 ng/ml rhIL-21 (Miltenyi Biotec, Germany) and 50 ng/ml rhIL-6 (R&D Systems, USA). Cells were incubated for 3 days at 37°C/5 %CO2. After incubation, supernatant was removed completely and 5 ml of new medium was added containing 10 ng/ml rhIL-15 (R&D Systems, USA), 50 ng/ml rhIL-6 (R&D Systems, USA) and 500 U/ml rhINFα (R&D Systems, USA). Cells were incubated for 4 days at 37°C/5 % CO2.

tag: donorid

tag: plateid

value: Fixed


title: PD67_PROCAP_A

city: Philadelphia

first: Gregory

last: Donahue

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

description:
sen.AB.Pooled.M.bw
sen.AB.Pooled.P.bw
Sen-not-Pro.hg19.bed

data_processing:
Index: an alignment index was constructed using BWA (command bwa index -a bwtsw) from a FASTA file which excluded alt/unassembled contigs and which included sequence U13369.1 (human rDNA) or BK000964.3 (mouse rDNA) as a separate contig so that tags aligning to it could be removed later.
Alignment: PRO-seq/cap data were aligned to the NCBI v37 (ENSEMBL release 75) assembly of the human genome (hg19) or the GRCm38 (ENSEMBL release 75) assembly of the mouse genome (mm10) using ProSeq 2.0 (Danko lab, https://github.com/Danko-Lab/proseq2.0) (component software versions used for this pipeline were bwa v0.7.10, fastx toolkit v0.0.14, samtools v1.1, seqtk v1.3-r115-dirty, cutadapt v2.10, prinseq-lite.pl v0.20.4, and bedtools v2.27.1-65-gc2af1e7-dirty). Paired-end data were aligned in mode -PE with parameters --RNA5=R1_5prime --RNA3=R2_5prime --ADAPT1=GATCGTCGGACTGTAGAACTCTGAAC --ADAPT2=TGGAATTCTCGGGTGCCAAGG.
Filtering: output BAM files were processed to remove unmapped reads using samtools view -F 4 and data were converted to BED format using bamToBed. Data sequenced on the Illumina NextSeq platform were then pooled across lanes. Tags containing adapter dimers were flagged in unaligned data using DimerRemover (https://github.com/royludo/DimerRemover) then removed from aligned data. All tags aligning to rDNA sequences were removed from aligned data using grep -v. Strands were reversed for single-end PRO-cap data. A and B replicates from PROCAP were pooled.
Track creation: bigWig files were made by splitting BED files by strand and then converting to a bedGraph using BEDtools genomeCoverageBed -bg. Tag pile-ups were multiplied by the RPM scalar then bedGraphs were sorted using UCSC Genome Browser tools bedSort and converted to bigWig with bedGraphToBigWig.
Peak calling: to call PRO-cap peaks in human IMR90 cells, we used HOMER's findPeaks method with -style tss.
Differential peaks: to obtain a stringent list of new PRO-cap peaks in the senescence condition, this initial list was further filtered to retain only those peaks that have a signal >4rpm (HOMER normalized tag counts) for human or >10rpm for mouse and >10-fold enrichment versus the opposite condition (e.g., sen > 10*pro) in an area-under-the-curve analysis. A pseudocount of 0.01 was added for fold change calculation to avoid division by zero. Any peak from this list that intersected (BedTools) with an RPM-passing peak from the other condition was removed.
Cryptic peak refinement: all peaks within 3.5 Kb of the nearest TSS (RefSeq hg19 or GENCODE hg19) and peaks which did not fall in the 3’ half of a gene body were also removed. The 3.5 Kb distance cutoff was chosen by assessing the distribution of distances of all ENCODE Broad chromHMM elements29 in state 1 or 2 (promoters) to the nearest RefSeq TSS; roughly 2/3 of elements are within 3.5 Kb. Peaks in PRO-cap replicate 1 were deemed to be reproduced if a peak in PRO-cap replicate 2 was identified within 5kb.
Assembly: hg19
Supplementary files format and content: BigWigs are strand-specific tracks of aligned PRO-seq/CAP tags (minus/M or plus/P with respect to the genomic reference strand).
Supplementary files format and content: BEDs are coordinates for condition-specific cryptic peaks from the PRO-CAP experiment.
Library strategy: PRO-CAP

growth_protocol: IMR90 cells, obtained from Coriell Institute for Medical Research, Camden, NJ, were grown in standard tissue culture medium (DMEM with 10% FBS and 1% penicillin/streptomycin; Invitrogen) at 3% oxygen.

extract_protocol:
PRO-seq/CAP was performed on proliferating and senescent IMR90 cells as described in Mahat, D.B. et al. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc 11, 1455-76 (2016). Briefly, ~12e6 trypsinized cells were permeabilized and stored in storage buffer (10mM Tris-HCl pH 8.0, 25% glycerol, 5mM MgCl2, 0.1mM EDTA, 5mM DTT) at -80°C until use. The permeabilized cells were then resuspended in 1X nuclear run-on reaction mix containing biotin-11-rCTP and unlabeled rATP, rGTP and rUTP for 3 minutes at 37°C. RNA was extracted, fragmented and subjected to biotin enrichment.
The enriched nascent RNA population was ligated to a 3’ adapter sequence and subjected to a second round of biotin enrichment. The 5’ ends of the RNA were decapped, phosphorylated, ligated to 5’ adapter and subjected to a third biotin enrichment. The RNA was then reverse transcribed and PCR amplified with appropriate index primers. The libraries were purified by extraction from an 8% polyacrylamide gel, quantified by Bioanalyzer (Agilent) and qPCR (Kapa Biosystems). The PRO-seq/CAP run was performed on the NextSeq 500 platform (Illumina).

treatment_protocol: Senescence was induced by replicative exhaustion. Briefly, for each passage, cells were washed with PBS, trypsinized at 37 °C for 5 minutes, and plated on fresh 10 cm plates at 0.5-1e6/plate or fresh 15cm plates at 1-2e6/plate. Cells were counted with a Countess automated cell counter (Life Technologies), and the numbers were recorded for growth curve generation. Cell viability record was maintained via Trypan Blue staining. Cells were maintained in dishes for 2 weeks to ensure growth termination/senescence at endpoint. Markers such as p16 expression and β-gal were tested to confirm senescence.

value: fibroblasts

value: senescent

tag: senescent

value: Yes


title: Fixed_D3_1588_Protein


title: Differentiating, low glucose, HA epitope, donor 2, input control

value: Low glucose


title: RRBS_TSC1_WT_late_passage

description: Bisulfite-converted DNA

growth_protocol: Late passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149).

treatment_protocol: no treatment


title: Fixed_D2_1587_Protein


title: ifos1_B


title: ifos1_A


title: Differentiating, low glucose, HA epitope, donor 2, ChIP-seq


title: ifos100_B

value: 100 ug/L ifosfamide


title: RRBS_TSC1_WT_late_passage_DNMT1i_7d_recovery_14d

description: Bisulfite-converted DNA

growth_protocol: Late passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DNMT1 inhibitor only  (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM to the medium. Subsequently, the cells were cultured for additionaly 14 days in TSC medium only (without additives) to allow recovery.


title: RRBS_TSC1_WT_late_passage_DNMT1i_7d

description: Bisulfite-converted DNA

growth_protocol: Late passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DNMT1 inhibitor only  (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM to the medium.


title: ERM_E

value: ERM, 1 ug/L


title: Live_D3_1585_Protein

value: Live


title: Differentiating, low glucose, HA epitope, donor 1, ChIP-seq


title: ERM_C


title: Differentiating, low glucose, DDX21 epitope, donor 2, input control


title: ERM_B


title: RRBS_TSC1_WT_late_passage_DMSO_7d

description: Bisulfite-converted DNA

growth_protocol: Late passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DMSO (Sigma, D2650) (1:1000).

value: DMSO


title: Live_D2_1580_Protein


title: Differentiating, low glucose, DDX21 epitope, donor 2, ChIP-seq


title: Differentiating, low glucose, DDX21 epitope, donor 1, input control


title: Live_D1_1579_Protein


title: RRBS_TSC1_WT_early_passage_DNMT1i_7d_recovery_14d

description: Bisulfite-converted DNA

growth_protocol: Early passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM. Subsequently, the cells were cultured for additionaly 14 days in TSC medium only (without additives) to allow recovery.

value: WT


title: Fixed_D2_1586_Protein


title: Differentiating, low glucose, DDX21 epitope, donor 1, ChIP-seq


title: cit1000_D

value: 1000 ug/L citalopram


title: Live_D1_1578_Protein


title: cit1000_C

value: larvae


title: RRBS_TSC1_WT_early_passage_DNMT1i_7d

description: Bisulfite-converted DNA

growth_protocol: Early passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: Differentiating, low glucose, HA epitope, donor 1, input control


title: Fixed_D3_1589_mRNA

data_processing:
Bcl2fasq software (illumina) was used for data-demultiplexing.
The Celseq2 pipeline or CITE-seq-count tool were used to create count tables of mRNA or protein libraries respectively. Standard settings were used with some minor modifications. The Celseq2 pipeline was run using settings [min_bc_quality = 10, cut_length = 50]. Additionally, the read names for read 1 and read 2 were swapped to allow compatibility of our barcode design with the pipeline. The CITE-seq-count settings were kept at default with exeption of [ max_error of Ab barcode = 1, umi_collpasing_dist = 1].
Genome_build: GRCh38
Supplementary_files_format_and_content: For the mRNA modality and protein modality count tables were generated called CITEseq_####.txt and COUNTS_####.tab. In rows one can find features (transcript or protein respectively) and in each column is one cell


title: cit1000_B


title: Live_D2_1584_Protein


title: RRBS_TSC1_WT_early_passage_DMSO_7d_recovery_14d

description: Bisulfite-converted DNA

growth_protocol: Early passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DMSO (Sigma, D2650) (1:1000). Subsequently, the cells were cultured for additionaly 14 days in TSC medium only (without additives) to allow recovery.


title: cit1000_A


title: Progenitor keratinocytes DDX21 shRNA-3 bio rep 2 tech rep 1

data_processing:
STAR (Love et al., 2014) (v. 2.7.8a) was used to map raw, paired-end, fastq files with the parameter “—quantMode TranscriptomeSAM”.
Transcriptome-aligned bam files were passed to the RSEM (Li and Dewey, 2011) (v. 1.3.3) program rsem-calculate-expression to generate read counts per transcript.
The featureCounts function from Rsubread (v. 2.0.1) was used to estimate reads per gene using the v.99 release of GRCh38 ENSEMBL.
Genome_build: GRCh38 ENSEMBL v.99 annotations
Supplementary_files_format_and_content: Rsubread estimated reads per gene

growth_protocol: Primary human keratinocytes were isolated from fresh surgically discarded newborn foreskin, and progenitor cells cultured in complete Keratinocyte-SFM (Life Technologies, #17005-142) and Medium 154 (Life Technologies #M-154-500). Cells were induced to differentiate by the addition of 1.2 mM calcium for 3 days at full confluence. Cells with low glucose and the corresponding normal glucose condition were maintained in glucose-free DMEM (Thermo Fishier, #11966025) supplied with SFM supplement (rEGF: Gibco, #10450-013; BPE: 13028-014), 1X Sodium Pyruvate (Gibco, 11360070) and a final concentration of 0.5 g/L (“low” glucose, Gibco, A2494001) or 4.5 g/L glucose (“normal” glucose) with adding 1.2 mM calcium during differentiation for 3 days. Organotypic regeneration of human epidermal tissue were performed as previously described (Truong et al., 2006). Biological replicates were performed in all cases using primary cells from at least 2 independent, unrelated donors.

extract_protocol:
RNA was extracted for sequencing using an RNeasy Plus kit (QIAGEN, 74136).
For poly(A)-primed RNA-seq, libraries were prepared with QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen, 015.96) following manufacturer’s protocol. RNA-seq was performed on a NovaSeq 6000, PE150, with ~30 M reads for each sample.

value: DDX21 shRNA-3


title: Fixed_D3_1588_mRNA


title: RRBS_TSC1_WT_early_passage_DMSO_7d

description: Bisulfite-converted DNA

growth_protocol: Early passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DMSO (Sigma, D2650) (1:1000).


title: Progenitor keratinocytes DDX21 shRNA-2 bio rep 2 tech rep 2

value: DDX21 shRNA-2


title: Fixed_D2_1587_mRNA


title: Live_D3_1582_Protein


title: RRBS_TSC1_WT_late_passage_DMSO_7d_recovery_14d

description: Bisulfite-converted DNA

growth_protocol: Late passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DMSO (Sigma, D2650) (1:1000). Subsequently, the cells were cultured for additionaly 14 days in TSC medium only (without additives) to allow recovery.


title: ERM_A


title: cit10_D

value: 10 ug/L citalopram


title: RRBS_TSC1_WT_early_passage

description: Bisulfite-converted DNA

growth_protocol: Early passage TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149).


title: Fixed_D2_1586_mRNA


title: cit10_C


title: Progenitor keratinocytes DDX21 shRNA-2 bio rep 2 tech rep 1


title: RRBS_TSC1_WT_EZH2i_5w_Rep2_recovery_3w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM. Subsequently, the cells were cultured for additionaly 21 days in TSC medium only (without additives) to allow recovery.


title: Progenitor keratinocytes control shRNA bio rep 2 tech rep 2

value: control shRNA


title: Live_D3_1585_mRNA


title: Progenitor keratinocytes DDX21 shRNA-3 bio rep 2 tech rep 2


title: RRBS_TSC1_WT_EZH2i_5w_Rep2_recovery_2w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM. Subsequently, the cells were cultured for additionaly 14 days in TSC medium only (without additives) to allow recovery.


title: Live_D2_1584_mRNA


title: cit10_A


title: Live_D3_1582_mRNA


title: RRBS_TSC1_WT_EZH2i_5w_Rep2_recovery_1w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM. Subsequently, the cells were cultured for additionaly 7 days in TSC medium only (without additives) to allow recovery.


title: 3D differentiated keratinocytes DDX21 shRNA-3 bio rep 2 tech rep 1

value: Organoid


title: 3D differentiated keratinocytes DDX21 shRNA-3 bio rep 2 tech rep 2


title: RRBS_TSC1_WT_EZH2i_5w_Rep2

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: Progenitor keratinocytes control shRNA bio rep 2 tech rep 1


title: cit10_B


title: Live_D2_1580_mRNA

growth_protocol: B-cells were pre-enriched using the RossetteSep Human B-cell Enrichment cocktail according manufacturer’s instructions (StemCell, USA). After Ficoll density gradient total B-cells were further isolated using the B cell isolation Kit II according manufacturer’s instructions (Miltenyi Biotec, Germany).


title: Live_D1_1579_mRNA


title: RRBS_TSC1_WT_EZH2i_5w_Rep1

description: Bisulfite-converted DNA

treatment_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: 3D differentiated keratinocytes control shRNA bio rep 2 tech rep 2


title: RRBS_TSC1_WT_EZH2i_5w_Rep2_recovery_4w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM. Subsequently, the cells were cultured for additionaly 28 days in TSC medium only (without additives) to allow recovery.


title: HEK 293 METTL8 overexpression mitoribosome profiling

city: Nashville

first: James

last: Marks

state: Tennessee

address: 638A Robinson Research Building

institute: NIAMS/NIH

description: mitoribosome footprints

data_processing:
Library strategy: Ribosome Profiling
Cutadapt
Remove ncRNA reads with Bowtie 1 and deduplication of reads
Alignment of reads to chrM transcriptome with Bowtie 1
P-site determination and construction of BigWig files through Plastid and wigToBigWig
Ribosome density analysis through in-house scripts
Genome_build: chrM trascriptome based on HG38
Supplementary_files_format_and_content: Each data file is a comparison of either a knockout or overexpression cell line compared to the control. Each row is a separate analyzable codon, ignoring regions around ND4-ND4L and ATP8-ATP6 overlap. Treatment and control density refer to the total number of reads mapping to a codon. Relative density is the total density at each codon normalized to the total density within a gene. Fold change is the ratio of treatment relative density over control relative density.

source_name: Cell Culture

growth_protocol: Cells were grown in T-75 flasks to 70% confluence in DMEM media.

extract_protocol:
Cells were washed once with ice cold PBS, flash frozen on liquid nitrogen and stored at -80C until lysis. 667 μL of 1.5x Lysis buffer (20 mM Tris–HCl pH 7.8, 100 mM KCl, 10 mM MgCl2, 1 mM DTT, 1% triton X-100, 0.1% NP40, 1× complete phosphatase and protease inhibitors) was added to each flask and cells were collected with a cell scraper while flasks thawed on an ice slurry. ~1 mL of lysate was transferred to a chilled 1.5 mL tube, gently passed through a 27 gauge needle 10 times, and spun at 5,000G for 10 minutes at 4C. Digestion was carried out by adding 1,500 U of MNase, 10 μL of SUPErase•In,  and CaCl2 to a final concentration of 5mM to 400 μL of lysate and incubating at 22C for 1 hour. Digestion was stopped by adding EGTA to a final concentration of 6 mM. Lysate was loaded onto a 5-30% sucrose gradient (20mM Tris–HCl pH 7.8, 100 mM KCl, 10 mM MgCl2, and 1 mM DTT and spun for 2.5 hrs at 40,000 rpm in an SW40 rotor. The 55S mitoribosome fraction was collected using a BioComp Fractionator. Footprints were isolated through SDS/hot phenol/chloroform extraction.
Following each step, footprints or PCR products were cleaned by either Zymo’s Oligo Clean and Concentrator or DNA Clean and Concentrator. Small RNAs were size selected using the Purelink miRNA isolation kit. 3’ ends were dephosphorylated with NEB Quick CIP and 3’ adapter ligation was carried out with Rnl2(1-249)K227Q ligase over night at 4°C. 3’ adapter: 5'-rAppNNTGACTGTGGAATTCTCGGGTGCCAAGG-L, where the underlined sequence is the library barcode. Barcoded footprint libraries were then combined and phosphorylated by PNK. Footprints were then ligated to a 5’ DNA-RNA adapter with RNA Ligase. 5'(aminolinker)GTTCAGAGTTCTACAGTCCGACGATCrNrNrNrN. Reverse transcription was carried out with SuperScript IV. RT Primer GCCTTGGCACCCGAGAATTCCA. Libraries were initially amplified by PCR using the RT primer and a short 5’ primer, CTTCAGAGTTCTACAGTCCGACGA. Footprints were size selected by a Pippen Prep, 3% C gel. Sizes of 74-94 were selected, corresponding to footprint sizes of 15-35. Libraries were then amplified with long indexing primers and sequenced.

treatment_protocol: METTL8 overexpression was induced with 2 μg/mL of doxycycline 24 hours prior to collection

value: HEK 293

value: METTL8 overexpression

tag: molecule subtype

value: Ribosome footprints


title: 3D differentiated keratinocytes DDX21 shRNA-2 bio rep 2 tech rep 1


title: HEK 293 METTL8 KO 12 mitoribosome profiling

description: mitoribosome footprints

value: METTL8 KO


title: Live_D1_1578_mRNA


title: RRBS_TSC1_WT_EZH2i_5w_Rep1_DNMT1i_1w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 1 week culture with the addition of DNMT1 inhibitor only  (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM to the medium.


title: RRBS_TSC1_WT_EZH2i_5w_Rep1_DNMT1i_1w_recovery_2w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 1 week culture with the addition of DNMT1 inhibitor only  (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM to the medium. Subsequently, the cells were cultured for additionaly 14 days in TSC medium only (without additives) to allow recovery.


title: 3D differentiated keratinocytes control shRNA bio rep 2 tech rep 1


title: HEK 293 METTL8 KO 7 mitoribosome profiling

description: mitoribosome footprints


title: 2D differentiated keratinocytes DDX21 shRNA-3 bio rep 2 tech rep 2


title: RRBS_TSC1_WT_EZH2i_4w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 4 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: HEK 293 WT mitoribosome profiling

description: mitoribosome footprints


title: RRBS_TSC1_WT_EZH2i_5w_Rep1_DMSO_1w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 1 week culture with the addition of DMSO (Sigma, D2650) to the medium (1:1000) instead of EZH2i.


title: 2D differentiated keratinocytes DDX21 shRNA-3 bio rep 2 tech rep 1


title: RRBS_TSC1_WT_EZH2i_5w_Rep1_DMSO_1w_recovery_2w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 1 week culture with the addition of DMSO (Sigma, D2650) to the medium (1:1000) instead of EZH2i. Subsequently, the cells were cultured for additionaly 14 days in TSC medium only (without additives) to allow recovery.


title: RRBS_TSC1_WT_EZH2i_3w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 3 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: CD70_PROCAP

data_processing:
Trimming/alignment: PRO-seq/CAP data were aligned to the NCBI v37 assembly of the human genome (hg19) using ProSeqMapper (https://github.com/Danko-Lab/tutorials/blob/master/PRO-seq.md) with default parameters and a BWA index.
Tracks: tracks for visualization were created using output BAM files from ProSeqMapper and UCSC Genome Browser Tools' bedGraphToBigWig.
Genome_build: hg19
Supplementary_files_format_and_content: BigWigs are strand-specific tracks of aligned PRO-seq/CAP tags (minus/M or plus/P with respect to the genomic reference strand).

source_name: IMR90 fibroblasts

growth_protocol: IMR90 cells, obtained from Coriell Institute for Medical Research, Camden, NJ, were grown in standard tissue culture medium (DMEM with 10% FBS and 1% penicillin/streptomycin; Invitrogen) at 3% oxygen.

extract_protocol:
PRO-CAP was performed as described in Mahat, D.B. et al. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc 11, 1455-76 (2016). Briefly, ~12e6 trypsinized cells were permeabilized and stored in storage buffer (10mM Tris-HCl pH 8.0, 25% glycerol, 5mM MgCl2, 0.1mM EDTA, 5mM DTT) at -80°C until use. The permeabilized cells were then resuspended in 1X nuclear run-on reaction mix containing biotin-11-rCTP and unlabeled rATP, rGTP and rUTP for 3 minutes at 37°C. RNA was extracted, fragmented and subjected to biotin enrichment.
The enriched nascent RNA population was ligated to a 3’ adapter sequence and subjected to a second round of biotin enrichment. The 5’ ends of the RNA were decapped, phosphorylated, ligated to 5’ adapter and subjected to a third biotin enrichment. The RNA was then reverse transcribed and PCR amplified with appropriate index primers. The libraries were purified by extraction from an 8% polyacrylamide gel, quantified by Bioanalyzer (Agilent) and qPCR (Kapa Biosystems). The PRO-CAP run was performed on the NextSeq 500 platform (Illumina).
OTHER: PRO-CAP

treatment_protocol: For overexpression experiments, IMR90 cells were infected with lentiviral constructs designed against selected targets in medium containing 8ug/ml polybrene. After 24 hours, the cells were replaced with polybrene-free media and after another 24 hours, selection was initiated with addition of blasticidin. After selection was complete, the media was changed back to standard medium. Cells were selected with blasticidin.

tag: sequencing type

value: PRO-CAP

value: No


title: 2D differentiated keratinocytes DDX21 shRNA-2 bio rep 2 tech rep 2

growth_protocol: Primary human keratinocytes were isolated from fresh surgically discarded newborn foreskin, and progenitor cells cultured in complete Keratinocyte-SFM (Life Technologies, #17005-142) and Medium 154 (Life Technologies #M-154-500). Cells were induced to differentiate by the addition of 1.2 mM calcium for 3 days at full confluence. Cells with low glucose and the corresponding normal glucose condition were maintained in glucose-free DMEM (Thermo Fishier, #11966025) supplied with SFM supplement (rEGF: Gibco, #10450-013; BPE: 13028-014), 1X Sodium Pyruvate (Gibco, 11360070) and a final concentration of 0.5 g/L (“low” glucose, Gibco, A2494001) or 4.5 g/L glucose (“normal” glucose) with adding 1.2 mM calcium during differentiation for 3 days. Organotypic regeneration of human epidermal tissue were performed as previously described (Truong et al., 2006). Biological replicates were performed in all cases using primary cells from at least 2 independent, unrelated donors.


title: RRBS_TSC1_WT_EZH2i_2w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 2 weeks with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: 3D differentiated keratinocytes DDX21 shRNA-2 bio rep 2 tech rep 2


title: 2D differentiated keratinocytes DDX21 shRNA-2 bio rep 2 tech rep 1


title: RNA-seq d4G cell NK - rep3

city: Clayton

first: Yi-Cheng

last: Chang

state: VIC

address: 15 Innovation Walk

department: ARMI

country: Australia

institute: Monash University

data_processing:
Two NextSeq550 V2.5 High output runs using 19bp forward for index read and 72bp reverse reads.
The reads were demultiplexed and trimmed using tool, sabre.
The reads are aligned to the to the mm10 genome using STAR aligner.
The minimum gene read count is 10 and theminimum gene CPM is 2.
The differential expression analysis was performed using edgeR, and only the genes with FDR < 0.05 would be considered as Differential expression genes (DEGs).
Genome_build: mm10
Supplementary_files_format_and_content: Raw gene counts for every gene and every sample
Supplementary_files_format_and_content: Differential expression genes (DEGs) in Nr6a1 mutants, relative to wildtpyes

source_name: ESC-derived differentiated cells

growth_protocol: The cells were cultured in N2B27 media (Composition: 49.5% Advanced Dulbecco’s Modified Medium F-12  (Gibco, 12634028); 49% Neurobasal medium (Gibco, 21103049); 0.5% N2-supplement (Gibco, 17502001); 1% B27-supplement (Gibco, 17504044)) supplied with 1x Glutamax (Gibco, 17504044), 40 µg/ml BSA Fraction V (Gibco, 15260037) and 100 mM 2-Mercaptoethanol (Gibco, 21985-023)

extract_protocol:
The RNA was extracted using Nucleospin RNA kit (Macherey-Nagel, 740955)
Novel RNA-Seq pipeline (Illumina Version20/02/2020). Briefly index is added during initial pA priming and pooled samples amplified using template switching oligo. P5 is added by PCR and P7 by Nextera transposase.

treatment_protocol: d2 cells (ESCs treated with FGF2 for two days) were treated with CHIR to activate Wnt signal and NMP identity. d3 cells (NMP) were then cultured in the same condition for one more day (d4) or treated with Gdf11 for one day (d4G)

tag: strain

value: ES-derived NMP differentiation - d4G

value: d3 cells treated with FGF2, CHIR and Gdf11 for 24h


title: CD27_PROCAP


title: BATF_PROCAP


title: RRBS_TSC1_WT_EZH2i_1w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 1 week with the addition of EZH2 inhibitor; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_7d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 7 days recovery without addition of DNMT1i.


title: 2D differentiated keratinocytes control shRNA bio rep 2 tech rep 2


title: 2D differentiated keratinocytes control shRNA bio rep 2 tech rep 1


title: RNA-seq d4G cell NK - rep2


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_6d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 6 days recovery without addition of DNMT1i.


title: Progenitor keratinocytes DDX21 shRNA-3 bio rep 1 tech rep 2


title: RNA-seq d4G cell NK - rep1


title: ATAC-seq.PD80.Sen.R1

data_processing:
Alignment: ATAC-seq paired end alignment was performed using bowtie2 on hg19 keeping only uniquely aligned tags (-k 1), 1 mismatch in the seed (-N 1) and fragment size not larger than 2000bp (-X 2000). SAM files were filtered by discarding fragments where a segment was unmapped (-F 4). Reads mapping to chrM were removed.
Pooling: proliferating and senescent replicate samples were pooled and then the proliferating sample was downsampled to have an equal number of tags to the senescent sample (python random module, sample() function).
Tracks: tracks for visualization were created using BEDtools genomeCoverageBed -bg and UCSC Genome Browser Tools' bedGraphToBigWig after adjusting for library size.
Genome_build: hg19
Supplementary_files_format_and_content: BigWigs are tracks of aligned ATAC-seq tag density after RPM adjustment.

extract_protocol:
To create ATAC-seq libraries the protocol from the Greenleaf Lab (Buenrostro, J.D., Wu, B., Chang, H.Y. & Greenleaf, W.J. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol 109, 21 29 1-9 (2015)) was employed. 100,000 proliferating or senescent IMR90 cells were used to generate libraries in two biological samples each.
The ATAC-seq run was performed on the NextSeq 500 platform (Illumina).

tag: population doubling


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_5d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 5 days recovery without addition of DNMT1i.


title: RNA-seq d4G cell WT - rep3

value: wildtype


title: ATAC-seq.PD35.Pro.R1


title: ATAC-seq.PD43.Pro.R2


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_4d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 4 days recovery without addition of DNMT1i.

value: WT


title: Progenitor keratinocytes DDX21 shRNA-2 bio rep 1 tech rep 2


title: RNA-seq d4G cell WT - rep2


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_2d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 2 days recovery without addition of DNMT1i.


title: RNA-seq d4G cell WT - rep1


title: Progenitor keratinocytes control shRNA bio rep 1 tech rep 2


title: ATAC-seq.PD80.Sen.R2


title: RNA-seq d4 cell NK - rep3

value: ES-derived NMP differentiation - d4

value: d3 cells treated with FGF2 and CHIR for 24h


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_3d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 3 days recovery without addition of DNMT1i.


title: Input.Pro.Rep4.PD26

data_processing:
Alignment: ChIP-seq alignment was performed using bowtie2 (v2.1.0) on hg19 keeping only alignments with 1 mismatch in the seed (-N 1).
Filtering: SAMtools (v0.1.19) was used to filter unmapped and poor quality alignments (samtools view -F 4 -q 10) while duplicate reads were also removed (rmdup) and data were converted to BED format using UCSC Genome Browser tools' bamToBed. Mitochondrial chromosome reads were removed with UNIX grep and BEDtools intersect was used to remove tags overlapping with ENCODE blacklisted regions.
Tracks: where possible, replicate data were pooled and tracks for visualization were created using BEDtools genomeCoverageBed -bg and UCSC Genome Browser Tools' bedGraphToBigWig after adjusting for library size; matching control samples were subtracted.
Genome_build: hg19
Supplementary_files_format_and_content: BigWigs are tracks of aligned ChIP-seq tag density after RPM adjustment and either H3 (for H3K79me1, H3K79me2, H3K36me3) or input (for H2A.z) subtraction.

extract_protocol:
ChIP was performed as described previously from ~12E6 cells (Shah, P.P. et al. Lamin B1 depletion in senescent cells triggers large-scale changes in gene expression and the chromatin landscape. Genes Dev 27, 1787-99 (2013).). Briefly, cross-linked cells were lysed, and the chromatin was sheared to an average size of <500 bp using a Covaris S220 Ultrasonicator. 2ug of sonicated chromatin was used per immunoprecipitation reaction (see Table S3 for a list of antibodies), and 10% of the amount was saved as input. Immunoprecipitation was performed using protein A Dynabeads (Life Technologies).
For ChIP-seq, 1ng of DNA from immunoprecipitation and input was used to prepare libraries with the NEBNext Ultra II kit (New England Biolabs). The libraries were checked for quality and quantity by Bioanalyzer (Agilent) and qPCR (Kapa Biosystems) respectively. Multiplexed libraries were sequenced on a single lane of the NextSeq 500 platform (Illumina).

tag: histone ptm

value: Input

tag: antibody, cat #

value: proliferating cells

tag: matching background


title: Input.Sen.Rep4.PD79

tag: condition

value: replicative senescent cells


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_28d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 28 days recovery without addition of DNMT1i.

tag: treatment


title: H2A.z.Sen.Rep2.PD79

value: Abcam ab4174


title: 3D differentiated keratinocytes DDX21 shRNA-3 bio rep 1 tech rep 2

value: DDX21 shRNA-3

tag: donor

tag: growth

value: Organoid


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_14d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 14 days recovery without addition of DNMT1i.


title: Input.Sen.Rep3.PD76

data_processing:
Alignment: ChIP-seq alignment was performed using bowtie2 (v2.1.0) on hg19 keeping only alignments with 1 mismatch in the seed (-N 1).
Filtering: SAMtools (v0.1.19) was used to filter unmapped and poor quality alignments (samtools view -F 4 -q 10) while duplicate reads were also removed (rmdup) and data were converted to BED format using UCSC Genome Browser tools' bamToBed. Mitochondrial chromosome reads were removed with UNIX grep and BEDtools intersect was used to remove tags overlapping with ENCODE blacklisted regions.
Tracks: tracks for visualization were created using BEDtools genomeCoverageBed -bg and UCSC Genome Browser Tools' bedGraphToBigWig after adjusting for library size; matching control samples were subtracted.
Genome_build: hg19
Supplementary_files_format_and_content: BigWigs are tracks of aligned ChIP-seq tag density after RPM adjustment and either H3 (for H3K79me1, H3K79me2, H3K36me3) or input (for H2A.z) subtraction.

tag: cell line/type

value: IMR90 fibroblasts


title: Progenitor keratinocytes DDX21 shRNA-3 bio rep 1 tech rep 1


title: RNA-seq d4 cell WT - rep3

tag: cell type


title: Input.Sen.Rep2.PD76


title: RRBS_TSC1_WT_DNMT1i_7d_recovery_1d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM, followed by 1 day recovery without addition of DNMT1i.


title: 3D differentiated keratinocytes DDX21 shRNA-3 bio rep 1 tech rep 1


title: H2A.z.Pro.Rep2.PD26


title: 3D differentiated keratinocytes DDX21 shRNA-2 bio rep 1 tech rep 2

value: DDX21 shRNA-2


title: RRBS_TSC1_WT_DNMT1i_7d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: Progenitor keratinocytes DDX21 shRNA-2 bio rep 1 tech rep 1


title: RNA-seq d4 cell WT - rep2

tag: genotype


title: 3D differentiated keratinocytes DDX21 shRNA-2 bio rep 1 tech rep 1


title: RNA-seq d4 cell WT - rep1


title: Input.Sen.Rep1.PD76


title: RRBS_TSC1_WT_DNMT1i_6d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 6 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: RNA-seq d4 cell NK - rep2


title: 3D differentiated keratinocytes control shRNA bio rep 1 tech rep 2

value: control shRNA


title: RRBS_TSC1_WT_DNMT1i_5d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: RNA-seq d4 cell NK - rep1


title: RNA-seq d3 cell NK - rep3

value: ES-derived NMP differentiation - d3

value: d2 cells treated with FGF2 and CHIR for 24h


title: H3.Sen.Rep3.PD76

growth_protocol: IMR90 cells, obtained from Coriell Institute for Medical Research, Camden, NJ, were grown in standard tissue culture medium (DMEM with 10% FBS and 1% penicillin/streptomycin; Invitrogen) at 3% oxygen.

value: Abcam ab1791


title: RRBS_TSC1_WT_DNMT1i_4d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 4 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: 3D differentiated keratinocytes control shRNA bio rep 1 tech rep 1

growth_protocol: Primary human keratinocytes were isolated from fresh surgically discarded newborn foreskin, and progenitor cells cultured in complete Keratinocyte-SFM (Life Technologies, #17005-142) and Medium 154 (Life Technologies #M-154-500). Cells were induced to differentiate by the addition of 1.2 mM calcium for 3 days at full confluence. Cells with low glucose and the corresponding normal glucose condition were maintained in glucose-free DMEM (Thermo Fishier, #11966025) supplied with SFM supplement (rEGF: Gibco, #10450-013; BPE: 13028-014), 1X Sodium Pyruvate (Gibco, 11360070) and a final concentration of 0.5 g/L (“low” glucose, Gibco, A2494001) or 4.5 g/L glucose (“normal” glucose) with adding 1.2 mM calcium during differentiation for 3 days. Organotypic regeneration of human epidermal tissue were performed as previously described (Truong et al., 2006). Biological replicates were performed in all cases using primary cells from at least 2 independent, unrelated donors.


title: RNA-seq d3 cell NK - rep2

growth_protocol: The cells were cultured in N2B27 media (Composition: 49.5% Advanced Dulbecco’s Modified Medium F-12  (Gibco, 12634028); 49% Neurobasal medium (Gibco, 21103049); 0.5% N2-supplement (Gibco, 17502001); 1% B27-supplement (Gibco, 17504044)) supplied with 1x Glutamax (Gibco, 17504044), 40 µg/ml BSA Fraction V (Gibco, 15260037) and 100 mM 2-Mercaptoethanol (Gibco, 21985-023)


title: RRBS_TSC1_WT_DNMT1i_3d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 3 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: RNA-seq d3 cell NK - rep1


title: 2D differentiated keratinocytes DDX21 shRNA-3 bio rep 1 tech rep 2


title: H3.Sen.Rep1.PD76


title: RRBS_TSC1_WT_DNMT1i_2d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 2 days with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: 2D differentiated keratinocytes DDX21 shRNA-3 bio rep 1 tech rep 1


title: RRBS_TSC1_WT_DNMT1i_1d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 1 day with the addition of Dnmt1 inhibitor (GSK-3484862; dissolved in DMSO to 1 mM) at a final concentration of 1 µM.


title: 2D differentiated keratinocytes DDX21 shRNA-2 bio rep 1 tech rep 2


title: H3K36me3.Sen.Rep2.PD74

value: Active Motif 61101


title: RNA-seq d3 cell WT - rep1

value: wildtype


title: 2D differentiated keratinocytes DDX21 shRNA-2 bio rep 1 tech rep 1


title: H3K36me3.Sen.Rep1.PD76


title: H2A.z.Sen.Rep1.PD76


title: RNA-seq d3 cell WT - rep2


title: RRBS_TSC1_WT_DMSO_7d_recovery_7d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium. After 7 days, cells were cultured further 7 days without addition of DMSO for recovery.

value: WT

value: DMSO


title: H3K79me2.Sen.Rep1.PD76

value: Abcam ab3594


title: RNA-seq d2.5 cell NK - rep3

value: ES-derived NMP differentiation - d2.5

value: d2 cells treated with FGF2 and CHIR for 12h


title: Progenitor keratinocytes control shRNA bio rep 1 tech rep 1


title: H3.Sen.Rep2.PD74

value: replicative senescent cells


title: RRBS_TSC1_WT_DMSO_7d_recovery_14d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium. After 7 days, cells were cultured further 14 days without addition of DMSO for recovery.


title: RNA-seq d2.5 cell NK - rep2


title: RRBS_TSC1_WT_DMSO_7d_recovery_28d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium. After 7 days, cells were cultured further 28 days without addition of DMSO for recovery.


title: 2D differentiated keratinocytes control shRNA bio rep 1 tech rep 1


title: H3K79me1.Sen.Rep1.PD76

value: Abcam ab2886


title: 2D differentiated keratinocytes control shRNA bio rep 1 tech rep 2


title: RNA-seq d2.5 cell NK - rep1


title: Input.Pro.Rep3.PD25

value: Input

value: proliferating cells


title: alg-2 IR 3

city: Cologne

first: Bjoern

last: Schumacher

state: NRW

address: Joseph-Stelzmann-Strasse 26

department: Institute for Genome Stability in Ageing and Disease

institute: CECAD Cologne

data_processing:
microRNA-Seq data were processed through the QuickNGS analysis pipeline (Wagle et al., 2015)
Reads were analyzed using mirDeep2
Differential gene expression was analyzed using the DESeq2 package
Genome_build: Caenorhabditis elegans – miRBase version 21
Supplementary_files_format_and_content: CSV files include  FPKM values of each Sample.

organism: Caenorhabditis elegans

source_name: whole animal

growth_protocol: Animals were grown at 20°C until day-1 adult stage on petri dishes containing NGM-agar seeded with E. coli OP50 as food source according to the standard protocol (Brenner, 1974)

extract_protocol:
Worms were lysed in Trizol reagent. Total RNA was isolated using the mirVana miRNA isolation kit (ThermoFisher).
Library layout: single read 50bp; Library source: small RNA libraries Ilumina TruSeq, strand specific; Library selection: miRNA selected due to size distribution

treatment_protocol: 1000 WT and alg-2(ok304) mutant day-1 adults were exposed to 90 Gy of γ-irradiation or left untreated to serve as controls. Afterwards, animals were allowed to recover for 2.5 hours at 20°C before proceeding with RNA extraction. Experiment was done in biological triplicates.

tag: strain/genotype

value: N2 Bristol

value: Ionizing radiation (90 Gy)

tag: age

value: day-1 adult


title: RRBS_TSC1_WT_DMSO_5w_Rep1

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 5 weeks with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium


title: alg-2 IR 2


title: RNA-seq d3 cell WT - rep3

value: ES-derived NMP differentiation - d3


title: alg-2 IR 1


title: RRBS_TSC1_WT_DMSO_4w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 4 weeks with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium


title: RNA-seq d2.5 cell WT - rep2


title: RNA-seq d2.5 cell WT - rep3


title: Input.Pro.Rep1.PD23

value: IMR90 fibroblasts


title: RRBS_TSC1_WT_DMSO_3w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 3 weeks with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium


title: Input.Pro.Rep2.PD25


title: RNA-seq d2.5 cell WT - rep1


title: RRBS_TSC1_WT_DMSO_2w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 2 weeks with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium


title: alg-2 Cont 3

value: Untreated


title: H3.Pro.Rep3.PD25

value: Abcam ab1791


title: alg-2 Cont 2


title: H3.Pro.Rep2.PD25


title: RNA-seq d2 cell NK - rep3

value: ES-derived NMP differentiation - d2

value: ESCs treated with FGF2 for 48h


title: alg-2 Cont 1


title: RRBS_TSC1_WT_DMSO_7d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 7 days with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium

value: WT

value: DMSO


title: H3.Pro.Rep1.PD23


title: RNA-seq d2 cell NK - rep2


title: RRBS_TSC1_WT_DMSO_1w

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149) for 1 week with the addition of DMSO (Sigma, D2650) (1:1000) to the culture medium


title: RRBS_ESC_WT_single_cell_sorted_cl9

description: Bisulfite-converted DNA

source_name: Embryonic stem cells

growth_protocol: ESC (clone 9) wild type cells were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

treatment_protocol: ESC (clone 9) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.


title: RRBS_ESC_WT_single_cell_sorted_cl8

description: Bisulfite-converted DNA

growth_protocol: ESC (clone 8) wild type cells were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

treatment_protocol: ESC (clone 8) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.


title: N2 IR 3

value: wild type


title: H3K36me3.Pro.Rep2.PD25

growth_protocol: IMR90 cells, obtained from Coriell Institute for Medical Research, Camden, NJ, were grown in standard tissue culture medium (DMEM with 10% FBS and 1% penicillin/streptomycin; Invitrogen) at 3% oxygen.

value: Active Motif 61101


title: RNA-seq d2 cell WT - rep3

growth_protocol: The cells were cultured in N2B27 media (Composition: 49.5% Advanced Dulbecco’s Modified Medium F-12  (Gibco, 12634028); 49% Neurobasal medium (Gibco, 21103049); 0.5% N2-supplement (Gibco, 17502001); 1% B27-supplement (Gibco, 17504044)) supplied with 1x Glutamax (Gibco, 17504044), 40 µg/ml BSA Fraction V (Gibco, 15260037) and 100 mM 2-Mercaptoethanol (Gibco, 21985-023)

value: wildtype


title: RRBS_ESC_WT_single_cell_sorted_cl7

description: Bisulfite-converted DNA

growth_protocol: ESC (clone 7) wild type cells were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

treatment_protocol: ESC (clone 7) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.


title: N2 IR 2


title: N2 IR 1


title: RRBS_ESC_WT_single_cell_sorted_cl6

description: Bisulfite-converted DNA

growth_protocol: ESC (clone 6) wild type cells were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

treatment_protocol: ESC (clone 6) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.


title: RNA-seq d2 cell WT - rep2


title: N2 Cont 3


title: H3K79me2.Pro.Rep1.PD25

value: Abcam ab3594


title: H3K79me1.Pro.Rep1.PD25

value: Abcam ab2886


title: RRBS_ESC_WT_single_cell_sorted_cl5

description: Bisulfite-converted DNA

growth_protocol: ESC (clone 5) wild type cells were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

treatment_protocol: ESC (clone 5) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.


title: H3K36me3.Pro.Rep1.PD23

value: proliferating cells


title: Nanopore_TSC1_WT

data_processing:
Nanopore data was processed using Nanopype (v1.1.0, base calling: guppy v4.0.11 with r9.4.1 high-accuracy configuration; alignment: minimap2 v2.10 with -ax map-ont -L --MD; methylation calling: nanopolish v0.13.2; reference genome: mm10)
Only methylation calls with an absolute likelihood of at least 2.5 were used for downstream analyses.
Assembly: mm10
Supplementary files format and content: Bigwig file containing methylation rates for CpGs covered by at least 10 and at most 150 reads: <chr> <start> <end> <methylation rate>
Library strategy: Nanopore sequencing

growth_protocol: TSCs (TSC line 1) were cultured in TSC medium (RPMI+GlutaMAX (Thermo, #61870044), 20 % fetal bovine serum (PAN, #P30-2602), 1 mM sodium pyruvate (Thermo, #11360070), 100 µM 2-mercaptoethanol (Thermo, #21985023) and 1X Penicillin/Streptomycin (Thermo, #15140122); 25 ng/ml FGF4 (R&D systems, #235-F4-025) and 1 µg/ml Heparin (Sigma, #H3149).

extract_protocol:
Ultra-high molecular weight DNA (UHMW DNA) was extracted with the Nanobind CBB big DNA kit (Circulomics, #NB-900-001-01) following the manufacturer's protocol with minor modifications.
We prepared an ultra-long nanopore sequencing library with a total of 35 µg of HMW DNA utilizing the Oxford Nanopore Technologies (ONT) sequencing library kit SQK-ULK001 following the manufacturer's protocol with minor modifications.

treatment_protocol: No treatment.

value: Trophoblast stem cells


title: N2 Cont 2

value: N2 Bristol


title: RNA-seq d2 cell WT - rep1


title: CK5_02428_3

city: 南昌

first: Jianmin

last: Bian

state: 江西 (Jiāngxī)

address: 志敏大道1101

country: China

institute: 江西农业大学

data_processing:
Illumina Hiseq4000 software used for basecalling.
The quality of sequenced reads were checked, and masked for low-complexity or low-quality sequence, then Hisat is used to compare the preprocessed Valid Data with reference genomes, and statistics are made separately according to the gene position information specified by the genome annotation file gtf.
Genome_build: Oryza_sativa.IRGSP-1.0.dna.toplevel.fa
Supplementary_files_format_and_content: 4_genes_fpkm_expression.txt: Matrix table with FPKM values for every gene and every sample.

organism: Oryza sativa Japonica Group

source_name: seedlings

growth_protocol: Thirty germinative seeds were placed in each dish, and the treatment groups (Cd) were treated with 200mg/L CdCl2 while the control groups (CK) with distilled water. Seedlings were maintained in the incubator at 28℃/25℃ for 16/8 h light/dark condition.

extract_protocol:
RNA was isolated using Trizol reagent Kit.
RNA libraries were prepared for sequencing using Illumina Hiseq4000 protocols.

treatment_protocol: After three days of cultivation, the seedlings of two rice varieties treated with cadmium and control were sampled respectively.

tag: developmental stage

value: germination stage

tag: tissue

value: seedlings

value: control


title: N2 Cont 1

value: Untreated

value: day-1 adult


title: TP309_panicle_mRNA_replicate2

city: Nanjing

first: Lili

last: Wang

address: Weigang No.1

institute: Nanjing Agricultural University

description: TP309_panicle_rep2 mRNA-seq

data_processing:
Low-quality reads had been eliminated.
All reads were mapped onto the O.sativa genome (MSU version 7.0) by Tophat.
Expression level were normalized through fragments per kilobase of exon model per million mapped reads (FPKM).
Genome_build: MSU version 7.0

source_name: panicle

growth_protocol: Rice plants grown in soil in Jiangsu province

extract_protocol:
RNA was extrated using TRIzol method.
1.Total RNA used DNaseI to digest DNA, and enriched mRNA using magnetic beads with Oligo(dT).  2.Adding reagent in Thermomixer to interrupt mRNA into short fragments.  3.The fragmented mRNA was used as template to synthesize the first strand of cDNA, and then synthesized the second strand.  4.Purified by using the kit, and repaired the adhesive termination, added the base "A" and adapter in the CDNA 3'end, then selected 200-300bp fragments and performed PCR amplification.   5.The libraries were checked with Agilent 2100 Bioanalyzer and ABI Real-Time PCR System, the qualified libraries were sequenced using sequencing platform.

value: panicle

tag: cultivar


title: H2A.z.Pro.Rep1.PD23

value: IMR90 fibroblasts

value: Abcam ab4174


title: RNA-seq d2 cell NK - rep1

tag: strain

value: ES-derived NMP differentiation - d2

value: ESCs treated with FGF2 for 48h


title: ATAC-seq.PD33.Pro.R3

data_processing:
Alignment: ATAC-seq paired end alignment was performed using bowtie2 on hg19 keeping only uniquely aligned tags (-k 1), 1 mismatch in the seed (-N 1) and fragment size not larger than 2000bp (-X 2000). SAM files were filtered by outputting all fragments where each segment properly aligned according to the aligner (-f 2) and discarding fragments where a segment was unmapped (-F 4).
Tracks: tracks for visualization were created using BEDtools genomeCoverageBed -bg and UCSC Genome Browser Tools' bedGraphToBigWig after adjusting for library size.
Genome_build: hg19
Supplementary_files_format_and_content: BigWigs are tracks of aligned ATAC-seq tag density after RPM adjustment.

extract_protocol:
To create ATAC-seq libraries the protocol from the Greenleaf Lab (Buenrostro, J.D., Wu, B., Chang, H.Y. & Greenleaf, W.J. ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide. Curr Protoc Mol Biol 109, 21 29 1-9 (2015)) was modified to replace the lysis buffer with a commercial buffer that reduces mitochondrial contamination. 100,000 proliferating or senescent IMR90 cells were used to generate libraries in two biological samples using 8mL transposase.
The ATAC-seq run was performed on the NextSeq 500 platform (Illumina).

tag: population doubling


title: RRBS_ESC_WT_single_cell_sorted_cl4

description: Bisulfite-converted DNA

data_processing:
Raw reads were subjected to adapter and quality trimming using cutadapt (version 2.4; parameters: --quality-cutoff 20 --overlap 5 --minimum-length 25; Illumina TruSeq adapter), followed by NuGEN diversity adapter trimming (https://github.com/nugentechnologies/NuMetRRBS).
The trimmed reads were aligned to the mouse genome (mm10) using BSMAP (version 2.90; parameters: -v 0.1 -s 12 -q 20 -w 100 -S 1 -u -R -D C-CGG).
Aligned reads were deduplicated based on unique molecular identifiers (UMIs) using NuDup (https://github.com/nugentechnologies/nudup; parameters: --start 6 --length 6). Methylation rates were called using mcall from the MOABS package (version 1.3.2; default parameters).
All analyses were restricted to autosomes and only CpGs covered by at least 10 and at maximum 150 reads were considered for downstream analyses.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig file containing methylation rates for CpGs covered by at least 10 and at most 150 reads: <chr> <start> <end> <methylation rate>

growth_protocol: ESC (clone 4) wild type cells were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

extract_protocol:
The DNA was extracted using the PureLink Genomic DNA Mini Kit (Thermo, #K182002) according to manufacturer’s instructions.
Concentration of genomic DNA (gDNA) was quantified using a Qubit 3.0 Fluorometer. RRBS was performed on 100 ng gDNA of each sample using the NuGen Ovation® RRBS Methyl-Seq System (Tecan, #0353) following the manufacturer’s recommendations with the following modifications (6). After the final repair step, the bisulfite conversion of DNA was conducted using the Qiagen EpiTect Fast Bisulfite Conversion kit (Qiagen, #59824) following the manufacturer’s recommendations, eluting the bisulfite converted DNA in 23 µl EB. Libraries were amplified with 12 cycles of PCR. Amplified library purification with Agencourt RNAclean XP beads (Beckman Coulter, #A63987) was performed twice (1X). The purified libraries were quality-assessed on an Agilent 4150 Tape-Station HS D1000 ScreenTape and sequenced for 100-bp single-end reads on the NovaSeq6000 (Illumina).

treatment_protocol: ESC (clone 4) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.

value: WT


title: RRBS_ESC_WT_single_cell_sorted_cl1

description: Bisulfite-converted DNA

growth_protocol: ESC (clone 1) wild type cells were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

treatment_protocol: ESC (clone 1) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.


title: CK5_02428_2


title: RRBS_ESC_WT_single_cell_sorted_cl2

description: Bisulfite-converted DNA

growth_protocol: ESC (clone 2) wild type cells were cultured in Knockout DMEM (Thermo, #10829018, 15% fetal bovine serum (PAN, #P30-2602), 1X GlutaMAX supplement (Thermo, #35050-038), 1X non-essential amino acids (Thermo, #11140-035), 100 µM 2-mercaptoethanol (Thermo, #21985023), 1X Penicillin/Streptomycin (Thermo, #15140122) and leukemia inhibitory factor (LIF)

treatment_protocol: ESC (clone 2) wild type cells were sorted as single cells onto gelatin and MEF-coated 96-well plates containing ESC medium. Cells were expanded and MEF-depleted for methylation analysis by RRBS.


title: ATAC-seq.PD75.Sen.R3

treatment_protocol: Senescence was induced by replicative exhaustion. Briefly, for each passage, cells were washed with PBS, trypsinized at 37 °C for 5 minutes, and plated on fresh 10 cm plates at 0.5-1e6/plate or fresh 15cm plates at 1-2e6/plate. Cells were counted with a Countess automated cell counter (Life Technologies), and the numbers were recorded for growth curve generation. Cell viability record was maintained via Trypan Blue staining. Cells were maintained in dishes for 2 weeks to ensure growth termination/senescence at endpoint. Markers such as p16 expression and β-gal were tested to confirm senescence.