title: MCF7_MR72NI_IP_rep1

description: MCF7 cells transfected with MR72 no intron, m6A IP sample, replicate 1, part of experiment 3


title: MINUTE_ESC_WT_H3K27me3_Pool1_Rep3

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: Quantitative ChIP of ESCs

library_source: genomic

data_processing:
Read pairs matching parts of the adaptor sequence (SBS3 or T7 promoter) in either read1 or read2 were removed using cutadapt v3.2.
Reads were demultiplexed using cutadapt v3.2 allowing only one mismatch per barcode and written into sample-specific FASTQ files used for subsequent mapping.
Sample-specific paired FASTQ files were mapped to the reference mm10 using bowtie2 v2.3.5.1 with --fast and --reorder parameter. Alignments were processed into sorted BAM files and replicates were pooled using samtools v1.10.
Duplicate reads are marked using UMI-sensitive deduplication tool je-suite (v2.0.RC) (https://github.com/gbcs-embl/Je/). Read pairs are marked as duplicates if their read1 (first-in-pair) sequences have the same UMI (allowing for 1 mismatch) and map to the same location in the genome. Blacklisted regions as downloaded from ENCODE were then removed from BAM files using bedtools v2.30.
Input coverage tracks with 1 bp resolution in bigwig format were generated from BAM files using deepTools v3.5.0 bamCoverage and scaled to a reads-per-genome-coverage of one (1xRPGC, also referred to as ‘1x normalization’) using the mm10 effective genome size. ChIP coverage tracks were generated from BAM files using deepTools (v3.5.0) bamCoverage. Quantitative scaling of the ChIP-Seq tracks amongst conditions within each pool was based on their Input-Normalized Mapped Read Count (INRC). INRC was calculated by dividing the number of unique reference-mapped reads by the respective number of Input reads: #mapped[ChIP] / #mapped[Input]. This essentially corrects for an uneven representation of barcodes in the Input. It has been previously shown that INRCs are proportional to the amount of epitope present in each condition (8). Reference condition (TSC WT) was scaled to 1x coverage (also termed Reads per Genome Coverage, RPGC). All other conditions within the same pool were scaled relative to the reference using the ratio of INRCs multiplied by the scaling factor determined for 1x normalization of the reference: ( #mapped[ChIP] / #mapped[Input] ) / ( #mapped[ChIP_Reference] / #mapped[Input_Reference] ) * scaling factor.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig files are scaled according to MINUTE quantification with respect to the reference of each pool (TSC1 WT, 1xRPGC)

molecule: genomic DNA

organism: Mus musculus

source_name: Embryonic stem cells

growth_protocol: Embryonic stem cells (ESC) cells were grown on gelatin/MEF-coated cell culture dishes containing ESC medium (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 1X leukemia inhibitory factor (LIF) (selfmade). Splitting was carried out every three days by rinsing the cells once with DPBS (Thermo, #14190144) before detaching the cells using TryplE (Gibco, #12604013). Before sample collection, ESCs were passaged at least one passage without MEFs to dilute out feeder cells. During this time, the cells were cultured in ESC medium containing 2X LIF.

extract_protocol:
Native cell pellets were lysed and digested with MNase to enrich for mononucleosome population. The digestion was quenched by EGTA-containing end-repair and ligation buffer, in which each sample was ligated to adaptor molecules carrying unique barcode. Ligation was quenched by EDTA-containing lysis dilution buffer, before combining all samples in one tube. After centrifugation, pool supernatant was recovered and aliquoted for individual ChIP. 2 x 10^6 cell-equivalent of pool supernatant was used for ChIP against each histone modification, with the anti-H3K4me3 (Millipore #04-745), anti-H3K27me3 (Cell Signaling #9733) and anti-H2AK119ub (Cell Signaling #8240) antibodies precoupled to Protein A magnetic beads.
After thorough washes, ChIP DNA was recovered with Proteinase K treatment and purified for linear amplification by in vitro transcription. The RNA product was then ligated to a pre-adenylated RNA 3’ adaptor (RA3), which served as a primer binding site for reverse transcription. The resulting cDNA was purified and used as a template for library PCR with barcoded primers compatible with Illumina sequencing platform. Typically, 1-2 x 10^5 cell equivalent of pool supernatant was used as Input, which is subjected to the same experimental workflow for library construction as the ChIP DNA. All nucleic-acid purification were carried out with AMPure SPRI size selection method (Beckman Coulter). Library size distribution was assessed by Agilent BioAnalyzer and were quantified by Qubit DNA high sensitivity assay before dilution for sequencing on a NovaSeq 6000 platform.

treatment_protocol: On the day of collection, ESCs were rinsed once with DPBS (Thermo, #14190144) and detached using TryplE (Gibco, #12604013). The cells were spun down for 4 min at 1000 rpm, counted and flash frozen on dry ice. The cell pellets were stored at -80°C.

tag: cell type

value: Embryonic stem cells

tag: condition

value: WT


title: MINUTE_ESC_WT_H3K4me3_Pool1_Rep1

description: Quantitative ChIP of ESCs


title: MPRE_IP_rep2

city: Rehovot

first: Anna

last: Uzonyi

address: 234 Herzl street

department: Department of Molecular Genetics

country: Israel

institute: Weizmann Institute of Science

description:
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq IP sample, rep2

library_source: transcriptomic

data_processing:
Fastq files were processed with a custom R script. Barcodes matching the planned barcode were counted for each sample. See Table S2 for the planned barcode sequences.
MPRE sequences based on mm9 and hg19, no alignment was performed
Csv file (BCcounts_raw.csv) containing raw barcode counts of each construct in the MPRE for input and IP of both biological relicates. Excel file (TableS2.xlsx) containing all planned construct sequences and description.

molecule: total RNA

organism: Homo sapiens

growth_protocol: HEK293T cells were cultured in DMEM (Gibco) supplemented with 10% FBS and 1% Penicillin and Streptomycin.

extract_protocol:
RNA was extracted with NucleoZOL (Macherey-Nagel)
RNA was poly-A selected with oligo dT-beads (Dynabeads® mRNA DIRECT™ Kit life tech). After mild fragmentation, samples were divided into input and IP sample, and IP samples were subjected to m6A immunoprecipitation. RNA from both the input and IP samples was reverse transcribed with a sequence specific RT primer to include the 10 nt unique barcode sequence of each construct in the MPRE. PCR amplicons of these fragments were generated.

treatment_protocol: 0.5 million HEK293T cells were seeded in each well of a 6-well plate. 24 hours later cells were transfected with 2 μg plasmid DNA and 8 ul home-made PEI reagent. Cells were harvested 24 hours post transfection. Each 3 wells of the 6-well plate were merged to form two replicates.

tag: sample type

value: human, mouse, yeast, synthetic

tag: cell line

value: human embryonic kidney

tag: treatment

value: MPRE plasmids transfection, MPRE contains ~12000 mouse, human, yeast and synthetic sequences


title: MPRE_IP_rep1

description:
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq IP sample, rep1


title: MINUTE_ESC_WT_H3K27me3_Pool1_Rep2

description: Quantitative ChIP of ESCs


title: MINUTE_ESC_WT_H2AK119ub1_Pool1_Rep3

description: Quantitative ChIP of ESCs


title: MCF7_MR73NI_IP_rep1

description: MCF7 cells transfected with MR73 no intron, m6A IP sample, replicate 1, part of experiment 3

data_processing:
Reads were mapped to hg19 genome assembly and the transfected plasmid sequences with STAR/2.7.9a (Dobin et al., 2013). Per-base read coverages were obtained with txtools (https://github.com/AngelCampos/txtools)
hg19 and the transfected plasmid (See TableS1 for sequence)
R data table (rds) file output from txtools https://github.com/AngelCampos/txtools. In addition, the TableS1.xlsx excel file contains the tranfected plasmid sequences.

molecule: polyA RNA

growth_protocol: MCF7 cells (ATCC) were maintained in DMEM (GIBCO) supplemented with 10% FBS and 1% Penicillin and Streptomycin.

extract_protocol:
RNA was extracted with BIO TRI RNA reagent (Bio-lab)
RNA was poly-A selected with oligo dT-beads (Dynabeads® mRNA DIRECT™ Kit life tech) and subjected to m6Aseq2 according to Dierks et al, 2021 Nature Methods.

treatment_protocol: Transfections were done with JetPrime (Polyplus) reagent, according to the supplier’s instructions. Typically, the cells were seeded in 6-cm dishes (Corning) and on the next day transfected with 1-2µg of plasmids. The cells were harvested after additional 24 hours by scraping and subjected to RNA extraction.

value: human cell line, plasmid with mouse sequence

value: human breast cancer

value: Plasmid transfection with an insert described in the 'description'. Insert sequences are in TableS1.xlsx.


title: MINUTE_ESC_WT_H2AK119ub1_Pool1_Rep2

description: Quantitative ChIP of ESCs


title: MPRE_input_rep1

description:
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq input sample, rep1


title: WGBS_TSC4_WT

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 clipped from both reads), followed by trimming of 10 and 5 nucleotides from the 5’ and 3’ end of the first read and 15 and 5 nucleotides from the 5’ and 3’ end of the second read.
The trimmed reads were aligned to the mouse genome (mm10) using BSMAP (version 2.90; parameters: -v 0.1 -s 16 -q 20 -w 100 -S 1 -u -R).
Duplicates were removed using the ‘MarkDuplicates’ command from GATK (version 4.1.4.1; --VALIDATION_STRINGENCY=LENIENT --REMOVE_DUPLICATES=true).
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 most 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>

source_name: Trophoblast stem cells

growth_protocol: TSCs (TSC line 4) 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. The DNA was sheared in Covaris micro TUBE AFA Fiber Pre-Slit Snap-Cap tubes (SKU: 520045) and cleaned up with the Zymo DNA Clean & Concentrator-5 kit (#D4013) following manufacturer’s guidelines. Sheared gDNA was bisulfite converted following manufacturer's guidelines with the EZ DNA Methylation-Gold Kit (Zymo #D5005), and libraries were prepared using the Accel-NGS Methyl-seq DNA library kit (Swift Biosciences, #30024-SWI). Libraries were cleaned using Agencourt AMPure XP beads (Beckman Coulter, #A63881), and the absence of adapters was confirmed on the Agilent TapeStation HS D5000. The final libraries were sequenced on a NovaSeq platform (Illumina) yielding 150 bp paired-end reads.

treatment_protocol: no treatment


title: VSMCs sample 12

city: Cincinnati

first: Albert

middle: Phillip

last: Owens III

state: OH

address: 231 Albert Sabin Way, CVC Room 3925, ML: 542

department: Internal Medicine

country: USA

institute: University of Cincinnati

data_processing:
All read mapping and analysis was performed using the CLC Genomics Workbench v20.0.2 (CLC Bio-Qiagen).
Reads were mapped using the following parameters:  min. read length fraction = 0.8, min. read similarity fraction = 0.8. mismatch cost = 2, insertion cost = 3, deletion cost = 3, Strand specific = both, Maximum number of hits for a read=10, Expression value= Total counts.
Assembly: hg19
Supplementary files format and content: transcript counts

source_name: Human aortic vascular smooth muscle cells

growth_protocol: HAVSMCs were purchased from LifeLine Cell Technology (Frederick, MD). Cells were plated directly into 6-well plates and cultured in the LifeLine Cell Technology VascuLife® SMC Medium containing 5 ng/mL rh FGF basic LifeFactor, 5 μg/mL rh Insulin LifeFactor, 50 μg/mL Ascorbic Acid LifeFactor, 10 mM L-Glutamine LifeFactor, 5 ng/mL rh EGF LifeFactor, 5% FBS LifeFactor, 30 mg/mL Gentamicin, and 15 μg/mL Amphotericin B (VascuLife® SMC Medium Complete Kit, LL-0014). Cells were maintained in a cell culture incubator at 37°C and 5% CO2.

extract_protocol:
RNA was isolated from cultured HAVSMCs with the commercially available NucleoSpin® RNA kit (Macherey-Nagel REF 740955) with several modifications. Briefly, media was removed and 300 μL QIAzol Lysis Reagent (QIAGEN Cat No. 79306) was added to each well of a 6-well plate. A cell scraper was used to scrape cells in QIAzol and cells from each well were collected into individual 1.5 mL tubes. Cells were vortexed and left to sit at room temperature for 5 minutes. To each tube, 60 μL of chloroform was added and cells were vortexed and left to sit for 2 – 3 minutes at room temperature. Cells were then centrifuged at 12,000 X g for 15 minutes at 4°C. The aqueous phase was removed to new tubes and 150 μL isopropyl alcohol was added to each. Following a 10-minute incubation at room temperature, each lysate was loaded onto a NucleoSpin® RNA Column and NucleoSpin® RNA protocol 5.1 was completed beginning at step five. Following elution, RNA was quantified with a NanoDrop™ 2000 Spectrophotometer (Thermo Scientific) and samples were stored at -80°C.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cultured HVSMCs were grown to 80% confluence and starved in DMEM/F12 media with HEPES (Corning REF # 10-092-CV), fetal bovine serum (1% vol/vol; Omega Scientific catalog FB-12), and penicillin/streptomycin (1% wt/vol) for 48 hours. Cells were then treated with either sterile PBS or TMAO (100 μM; Sigma-Aldrich, 317594) for 5 hours.

value: human aortic vascular smooth muscle cells (HAVSMCs)

tag: company of origin

value: Lifeline Cell Technology

value: TMAO (100μM)

tag: treatment time

tag: passages

value: 4 passages after arrival


title: MINUTE_ESC_WT_H3K27me3_Pool1_Rep1

description: Quantitative ChIP of ESCs


title: WGBS_TSC3_EEDKO

description: Bisulfite-converted DNA

growth_protocol: TSCs Eed KO clone A2 (background TSC line 3) was 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.

treatment_protocol: TSCs wildtype cells (TSC3 line) were transfected with Cas9/sgRNA system targeting the Eed gene.

value: EED KO


title: VSMCs sample 11


title: WGBS_TSC3_WT

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 3) 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: VSMCs sample 10


title: VSMCs sample 9


title: WGBS_TSC2_3BKO

description: Bisulfite-converted DNA

growth_protocol: TSCs Dnmt3b KO clone A12 (background TSC line 2) was 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 wildtype cells (TSC2 line) were transfected with Cas9/sgRNA system targeting the Dnmt3b gene.


title: WGBS_TSC2_WT

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: VSMCs sample 8


title: WGBS_TSC1_WT

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).


title: VSMCs sample 7

value: Saline


title: MCF7_MR72_IP_rep1

description: MCF7 cells transfected with MR72 with intron, m6A IP sample, replicate 1, part of experiment 3


title: WGBS_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: VSMCs sample 6


title: WGBS_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: VSMCs sample 5


title: WGBS_TSC1_RNF2KO

description: Bisulfite-converted DNA

growth_protocol: TSC Rnf2 KO clone H10 (background TSC1 line) was 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 wildtype cells (TSC1 line) were transfected with Cas9/sgRNA system targeting the Rnf2 gene.

value: RNF2 KO


title: MPRE_input_rep2

description:
RNA amplicon
HEK293T cells transfected with MPRE, m6Aseq input sample rep2


title: MINUTE_ESC_WT_H2AK119ub1_Pool1_Rep1

description: Quantitative ChIP of ESCs


title: RRBS_TSC2_WT_single_cell_sorted_cl9

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: TSCs (TSC2 line, clone 9) 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 (TSC2 line, 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: WGBS_TSC1_KDM2BKO

description: Bisulfite-converted DNA

growth_protocol: TSC Kdm2b KO clone C3 (background TSC1 line) was 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 wildtype cells (TSC1 line) were transfected with Cas9/sgRNA system targeting the Kdm2b gene.

value: KDM2B KO


title: RRBS_TSC2_WT_single_cell_sorted_cl8

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 8) 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 (TSC2 line, 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: DDX21 CLIP-seq, progenitor keratinocytes, normal glucose bio rep 3

city: Stanford

first: Douglas

last: Porter

state: CA

address: 269 Campus Drive

department: Dermatology

institute: Stanford

data_processing:
STAR (Love et al., 2014) (v. 2.7.1a) was used to map raw, paired-end, fastq files to the rDNA locus, then to a genome of repetitive elements, then to the genome.
Custom scripts were used for read processing (https://github.com/dfporter/easyclip-snakemake).
Reads were assigned to genes, separating exons and introns, using HTSeq (v. 0.13.5).
Genome_build: GRCh38 Gencode release 29
Supplementary_files_format_and_content: Unnormalized reads per gene or element.

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). HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) media (Thermo Fisher, #11995-065) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C with 5% CO2.  For infection in either cell type, the optimized viral titer was added to primary human keratinocytes together with polybrene (5 µg/mL). The next day, fresh media was added, and cells were selected using puromycin (1 µg/mL). Cells were infected with a HA-tagged DDX21 (WT or mutant) construct. Biological replicates were performed in keratinocytes using primary cells from at least 2 independent, unrelated donors.

extract_protocol:
Cells were harvested according to the easyCLIP protocol (PMID: 33692367) - lysis buffer was added to the plate, cell scraped into an eppendorf tube, and further lyzed by sonication.
Libraries were created using the easyCLIP protocol (PMID: 33692367), with modifications.
OTHER: CLIP-seq (easyCLIP)

treatment_protocol: Cells washed once with PBS on the petri dish, then UV cross-linked in a Stratalinker at 0.3 mJ/cm2.

value: normal glucose

tag: donor

tag: growth


title: DDX21 CLIP-seq, progenitor keratinocytes, normal glucose bio rep 1


title: DDX21 CLIP-seq, progenitor keratinocytes, normal glucose bio rep 2


title: DDX21 WT CLIP-seq, 293T, bio rep 3


title: Post-transplant donor cells Selp high from primary recipients, 4 month post transplant rep2

city: Groningen

first: Leonid

last: Bystrykh

email: l.bystrykh@umcg.nl, l.bystrykh@rug.nl

address: Ant.Deusinglaan 1

department: ERIBA

country: Netherlands

institute: UMCG

description: RNA isolated with RNAeasy plus micro kit (QIAGEN), cDNA made with SMART ultra-low Input kit v4 (TAKARA). 1 ng used to generate sequence libraries with Nextera TM XT Library Prep Kit (Illumina)

data_processing:
FASTQ files were quality-control checked using FastQC (0.11.9) and Picard (2.23.0).
Reads were mapped to reference mouse genome (GENCODE, GRCm38, M13) using STAR (2.7.0d) with standard arguments and --outFilterMatchNminOverLread 0.4 --outFilterScoreMinOverLread 0.4.
Genome_build: GENCODE, GRCm38, M13
Supplementary_files_format_and_content: reads per gene STAR txt files

source_name: LT-HSC from transplanted recipient (4 month post-transplant)

extract_protocol:
5000 LT-HSCs (LSK SLAM) from each sample were used for RNA isolation using RNeasy Plus Micro Kit (QIAGEN). cDNA was created using SMART Ultra-Low Input kit v4 (Takara).
1 ng cDNA was then used to generate Illumina sequencing libraries using the Nextera™ XT Library Prep kit (Illumina).

treatment_protocol:
BM cells were isolated by crushing from the tibia, femur, pelvis, sternum and spine and red blood cells were lysed with lysis buffer. For general LT-HSC isolation, lysed bone marrow was stained with lineage (Lin) markers antibodies (B220, CD3, Gr-1, Mac-1 and Ter-119) conjugated with Alexa 700, c-Kit-PE, Sca-1-BV421/Pacific Blue, CD48-Alexa 647 and CD150-PECy7 (all from Biolegend). After incubating with the antibody mix at 4℃ for 30 minutes, samples were washed, filtered and then LT-HSCs were isolated on MoFlo Astrios or XDP cell sorters (Beckman Coulter).
For Selp-lo and Selp-hi LT-HSC isolation, lysed bone marrow was stained with Lin-Alexa 700, c-Kit-FITC or BB515, Sca-BV421, CD48-Alexa 647, CD150-APCFire 750 (Biolegend), Selp-PE (eBioscience). Cells were isolated on MoFlo Astrios sorter.

value: Long-term hematopoietic cells

tag: strain

tag: gender

value: female


title: Post-transplant donor cells Selp high from primary recipients, 4 month post transplant rep3


title: RRBS_TSC2_WT_single_cell_sorted_cl7

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 7) 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 (TSC2 line, 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.

value: WT


title: Post-transplant donor cells Selp high from primary recipients, 4 month post transplant rep1


title: Post-transplant donor cells Selp low from primary recipients, 4 month post transplant rep3


title: RRBS_TSC2_WT_single_cell_sorted_cl6

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 6) 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 (TSC2 line, 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: Post-transplant donor cells Selp low from primary recipients, 4 month post transplant rep2


title: RRBS_TSC2_WT_single_cell_sorted_cl5

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 5) 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 (TSC2 line, 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: Post-transplant donor cells Selp low from primary recipients, 4 month post transplant rep1


title: DDX21 WT CLIP-seq, 293T, bio rep 1


title: DDX21 WT CLIP-seq, 293T, bio rep 2


title: Pre-transplant Selp high LT-HSCs from aged donor rep4

source_name: LT-HSC from old (>24mo) donor


title: DDX21 611R (dimerization region mutated) CLIP-seq, 293T, bio rep 3


title: Pre-transplant Selp high LT-HSCs from aged donor rep3


title: Pre-transplant Selp high LT-HSCs from aged donor rep2


title: RRBS_TSC2_WT_single_cell_sorted_cl3

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 3) 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 (TSC2 line, clone 3) 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: VSMCs sample 4

growth_protocol: HAVSMCs were purchased from LifeLine Cell Technology (Frederick, MD). Cells were plated directly into 6-well plates and cultured in the LifeLine Cell Technology VascuLife® SMC Medium containing 5 ng/mL rh FGF basic LifeFactor, 5 μg/mL rh Insulin LifeFactor, 50 μg/mL Ascorbic Acid LifeFactor, 10 mM L-Glutamine LifeFactor, 5 ng/mL rh EGF LifeFactor, 5% FBS LifeFactor, 30 mg/mL Gentamicin, and 15 μg/mL Amphotericin B (VascuLife® SMC Medium Complete Kit, LL-0014). Cells were maintained in a cell culture incubator at 37°C and 5% CO2.


title: DDX21 611R (dimerization region mutated) CLIP-seq, 293T, bio rep 2


title: Pre-transplant Selp high LT-HSCs from aged donor rep1


title: DDX21 611R (dimerization region mutated) CLIP-seq, 293T, bio rep 1


title: Pre-transplant Selp low LT-HSCs from aged donor rep3


title: Pre-transplant Selp low LT-HSCs from aged donor rep4


title: DDX21 K236R (ATP-binding mutant) CLIP-seq, 293T, bio rep 3


title: Pre-transplant Selp low LT-HSCs from aged donor rep2


title: RRBS_TSC2_WT_single_cell_sorted_cl1

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 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: TSCs (TSC2 line, 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: Pre-transplant Selp low LT-HSCs from aged donor rep1


title: RRBS_TSC2_WT_single_cell_sorted_cl12

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 12) 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 (TSC2 line, clone 12) 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 Cdx2P-Nr6a1 tailbud TG - rep2 [P13]

city: Clayton

first: Yi-Cheng

last: Chang

state: VIC

address: 15 Innovation Walk

department: ARMI

country: Australia

institute: Monash University

data_processing:
The reads were demultiplexed and trimmed using tool, sabre.
The reads are aligned to the reference genome using STAR aligner.
The transgenic positive samples with no significant up-regulated of Nr6a1 relative to wildtype were excluded for further analysis.
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: DEGs in the transgenic samples, relative to wildtpyes

source_name: E10.5 mouse tailbud

growth_protocol: Cdx2P:Nr6a1 transgenic embryos were generated by pronuclear injection, and the transgenic negative embryos were used as wildtype.

extract_protocol:
The RNA was extracted using Nucleospin RNA kit (Macherey-Nagel, 740955)
PolyA RNA-seq libraries prepared using Ilumina TruSeq Stranded mRNA Sample prep, Protocol 15031047 RevE Oct 2013

treatment_protocol: The dissected tailbud was immediately placed in RA1 lysis buffer (Macherey-Nagel) on dry ice and then stored at -80℃.

value: tailbud cells

tag: genotype


title: DDX21 K236R (ATP-binding mutant) CLIP-seq, 293T, bio rep 2


title: RNA-seq Cdx2P-Nr6a1 tailbud TG - rep1 [P4]


title: RNA-seq Cdx2P-Nr6a1 tailbud WT- rep4 [N24]

value: wildtype


title: RRBS_TSC2_WT_single_cell_sorted_cl11

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 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).

treatment_protocol: TSCs (TSC2 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.


title: RNA-seq Cdx2P-Nr6a1 tailbud WT- rep3 [N10]


title: DDX21 K236R (ATP-binding mutant) CLIP-seq, 293T, bio rep 1


title: RRBS_TSC2_WT_single_cell_sorted_cl10

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 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 (TSC2 line, clone 10) 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 Cdx2P-Nr6a1 tailbud WT- rep2 [N6]


title: RRBS_TSC2_WT_single_cell_sorted_cl4

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 4) 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 (TSC2 line, 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.


title: RNA-seq Cdx2P-Nr6a1 tailbud WT- rep1 [N5]


title: DDX21 K236G (ATP-binding mutant) CLIP-seq, 293T, bio rep 3


title: DDX21 K236G (ATP-binding mutant) CLIP-seq, 293T, bio rep 2


title: DDX21 K236G (ATP-binding mutant) CLIP-seq, 293T, bio rep 1


title: RRBS_TSC2_WT_DNMT1i_7d_recovery_7d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: RRBS_TSC2_WT_DNMT1i_7d_recovery_6d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: Young_Female_R2_PROCAP

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:
PROCAP1.YF.Peaks.10FC.Cryptic.bed
YF6.Pooled.M.bw
YF6.Pooled.P.bw

data_processing:
Library strategy: PRO-CAP
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.
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).
Supplementary_files_format_and_content: BEDs are coordinates for condition-specific cryptic peaks from the PRO-CAP experiment.

source_name: frozen liver

growth_protocol: C57/BL6J young mice were ordered from Jackson Laboratories, old mice were obtained from the National Institute on Aging rodent colony. Mice were fed ad libitum on a standard chow diet and handled following institutional regulations and guidelines. Both sexes were included in the study.

extract_protocol:
PRO-CAP (ChRO-CAP) was performed as described in Chu, T. et al. Chromatin run-on and sequencing maps the transcriptional regulatory landscape of glioblastoma multiforme. Nat Genet 50, 1553-1564 (2018); with ~250mg frozen liver tissue from young and old mice.
The PRO-CAP run was performed on the NextSeq 500 platform (Illumina).

treatment_protocol: Young mice were aged 2-4 months and old mice were aged 22-25 months

tag: age

value: 2-4 months

tag: Sex

value: Female

tag: tissue

value: frozen liver


title: Young_Female_R1_PROCAP

description:
PROCAP1.YF.Peaks.10FC.Cryptic.bed
YF5.Pooled.M.bw
YF5.Pooled.P.bw


title: RRBS_TSC2_WT_single_cell_sorted_cl2

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC2 line, clone 2) 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 (TSC2 line, 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: Young_Male_R1_PROCAP

description:
PROCAP1.YF.Peaks.10FC.Cryptic.bed
YM1.Pooled.M.bw
YM1.Pooled.P.bw

value: Male


title: RRBS_TSC2_WT_DNMT1i_7d_recovery_5d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: DDX21 611Del (dimerization region deleted) CLIP-seq, 293T, bio rep 3

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). HEK293T cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) media (Thermo Fisher, #11995-065) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin at 37°C with 5% CO2.  For infection in either cell type, the optimized viral titer was added to primary human keratinocytes together with polybrene (5 µg/mL). The next day, fresh media was added, and cells were selected using puromycin (1 µg/mL). Cells were infected with a HA-tagged DDX21 (WT or mutant) construct. Biological replicates were performed in keratinocytes using primary cells from at least 2 independent, unrelated donors.


title: Old_Male_R1_PROCAP

description:
PROCAP1.OF.Peaks.10FC.Cryptic.bed
OM1.Pooled.M.bw
OM1.Pooled.P.bw

value: 22-25 months


title: Old_Male_R2_PROCAP

description:
PROCAP1.OF.Peaks.10FC.Cryptic.bed
OM2.Pooled.M.bw
OM2.Pooled.P.bw


title: DDX21 611Del (dimerization region deleted) CLIP-seq, 293T, bio rep 2


title: RRBS_TSC2_WT_DNMT1i_7d_recovery_4d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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.


title: Old_Female_R2_PROCAP

description:
PROCAP1.OF.Peaks.10FC.Cryptic.bed
OF6.Pooled.M.bw
OF6.Pooled.P.bw


title: RRBS_TSC2_WT_DNMT1i_7d_recovery_3d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: Old_Female_R1_PROCAP

description:
PROCAP1.OF.Peaks.10FC.Cryptic.bed
OF5.Pooled.M.bw
OF5.Pooled.P.bw


title: DDX21 611Del (dimerization region deleted) CLIP-seq, 293T, bio rep 1


title: DDX21 CLIP-seq, differentiating keratinocytes, low glucose bio rep 3

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 harvested according to the easyCLIP protocol (PMID: 33692367) - lysis buffer was added to the plate, cell scraped into an eppendorf tube, and further lyzed by sonication.
Libraries were created using the easyCLIP protocol (PMID: 33692367).
OTHER: CLIP-seq (easyCLIP)

value: low glucose


title: DDX21 CLIP-seq, differentiating keratinocytes, low glucose bio rep 1


title: shDDX21 treated, differentiating keratinocytes, K256R DDX21 rescue, bio rep 2

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 exon using the v.104 release of GRCh38 ENSEMBL.
Genome_build: GRCh38 ENSEMBL v.104 annotations
Supplementary_files_format_and_content: Rsubread estimated reads per exon

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. Biological replicates were performed in all cases using primary cells from at least 2 independent, unrelated donors.

extract_protocol:
For RNA-seq, RNA was extracted for sequencing using an RNeasy Plus kit (QIAGEN, 74136).
For mRNA-seq splicing analysis, total RNA was extracted using the RNeasy Plus kit (QIAGEN, 74136), followed by poly(A) enrichment and library preparation using the NEB Ultra II (non-directional) kit (E6111). Libraries were sequenced on a NovaSeq 6000, PE150, with ~30 M reads for each sample.

treatment_protocol: shRNAs targeting DDX21 were designed based on Sigma sequences (https://www.sigmaaldrich.com/US/en/semi-configurators/shrna?activeLink=productSearch) and were cloned into pLKO.1-puro or pLKO.1-blasti plasmid (Addgene, #10878 and #26655). The targeting sequences are: shControl, 5’-TCCTAAGGTTAAGTCGCCCTCG-3’; shDDX21-2, 5’-GCATGAGGAATGGGATTGATA-3’. For infection, the optimized viral titer was added to primary human keratinocytes together with polybrene (5 µg/mL). The next day, fresh media was added, and cells were selected using puromycin (1 µg/mL).

value: DDX21 shRNA, K256R DDX21 rescue


title: DDX21 CLIP-seq, differentiating keratinocytes, normal glucose bio rep 3


title: Young_Male_R2_PROCAP

description:
PROCAP1.YF.Peaks.10FC.Cryptic.bed
YM2.Pooled.M.bw
YM2.Pooled.P.bw


title: shDDX21 treated, differentiating keratinocytes, DDX21 rescue, bio rep 2

value: DDX21 shRNA, DDX21 rescue


title: RRBS_TSC2_WT_DNMT1i_7d_recovery_1d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: DDX21 CLIP-seq, differentiating keratinocytes, normal glucose bio rep 2


title: DDX21 CLIP-seq, differentiating keratinocytes, normal glucose bio rep 1


title: shDDX21 treated, differentiating keratinocytes, GFP rescue, bio rep 2

value: DDX21 shRNA, GFP rescue


title: RRBS_TSC2_WT_DNMT1i_7d_recovery_14d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: D13_KBCG_REP6

city: London

first: JONATHAN

last: CARON

state: Greater London

address: New Hunt's House, Newcomen St

department: Cancer Studies

country: United Kingdom

institute: KING'S COLLEGE LONDON

data_processing:
Partek's build-in QC, Filtering (Bowtie 2), alignement (STAR 2.7.3a)
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes normalized gene counts for each Sample

source_name: BLOOD

growth_protocol: PBMCs cultured at 5x10^6 cells/ml in RPMI-1640 + 10% FBS

extract_protocol:
RNA was extracted from Vγ9Vδ2 T-cells using RNeasy Micro Kit (Qiagen) as per manufacturer’s  instructions.
mRNA library prepared using PROTOCOLNEBNext Ultra II kit, as per manufacturer’s  instructions.

treatment_protocol: Cells cultured with final concentrations of 10µM ZA, or 2x10^4 CFU BCG (Pasteur strain), both with 15ng/ml IL-2 in 200µl total volume in 96 well round bottomed plates.

value: BLOOD

value: GAMMA-DELTA T CELLS

value: HEAT-KILLED BCG


title: D13_KBCG_REP4


title: RRBS_TSC2_WT_DNMT1i_7d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: shControl treated, differentiating keratinocytes, GFP rescue, bio rep 2

value: control shRNA, GFP rescue


title: D13_KBCG_REP5


title: D13_KBCG_REP2


title: RRBS_TSC2_WT_DNMT1i_6d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: D13_KBCG_REP1


title: D13_KBCG_REP3


title: RRBS_TSC2_WT_DNMT1i_5d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: D13_BCG_REP6

value: Bacillus Calmette–Guérin


title: shDDX21 treated, differentiating keratinocytes, DDX21 rescue, bio rep 1


title: D13_BCG_REP5


title: shDDX21 treated, differentiating keratinocytes, K256R DDX21 rescue, bio rep 1


title: RRBS_TSC2_WT_DNMT1i_4d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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.

value: WT


title: D13_BCG_REP4


title: RRBS_TSC2_WT_DNMT1i_7d_recovery_2d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: shDDX21 treated, differentiating keratinocytes, GFP rescue, bio rep 1


title: DDX21 CLIP-seq, differentiating keratinocytes, low glucose bio rep 2


title: D13_BCG_REP3


title: D13_BCG_REP2


title: RRBS_TSC2_WT_DNMT1i_3d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: shControl treated, differentiating keratinocytes, GFP rescue, bio rep 1


title: Normal glucose media, differentiating keratinocytes, bio rep 2

treatment_protocol: shRNAs targeting DDX21 were designed based on Sigma sequences (https://www.sigmaaldrich.com/US/en/semi-configurators/shrna?activeLink=productSearch) and were cloned into pLKO.1-puro or pLKO.1-blasti plasmid (Addgene, #10878 and #26655). The targeting sequences are: shScramble, 5’-TCCTAAGGTTAAGTCGCCCTCG-3’; shDDX21-2, 5’-GCATGAGGAATGGGATTGATA-3’; shDDX21-3, 5’-CCCATATCTGAAGAAACTATT-3’. For infection, the optimized viral titer was added to primary human keratinocytes together with polybrene (5 µg/mL). The next day, fresh media was added, and cells were selected using puromycin (1 µg/mL).

value: DDX21 shRNA-3


title: D13_ZA_REP5

value: ZOLEDRONIC ACID


title: D13_BCG_REP1


title: RRBS_TSC2_WT_DNMT1i_2d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: RRBS_TSC2_WT_DNMT1i_1d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: D13_ZA_REP3


title: Progenitor keratinocytes, bio rep 2

value: DDX21 shRNA-2


title: RRBS_TSC2_WT_DMSO_7d

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC line 2) 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: DMSO


title: Low-glucose media, differentiating keratinocytes, bio rep 2


title: RRBS_TSC1_WT_single_cell_sorted_cl8

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 8) 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 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: RRBS_TSC1_WT_single_cell_sorted_cl7

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 7) 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 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: Normal glucose media, differentiating keratinocytes, bio rep 1


title: Low-glucose media, differentiating keratinocytes, bio rep 1

value: control shRNA


title: D13_IL2_REP4


title: D13_ZA_REP4


title: D13_ZA_REP1


title: D13_IL2_REP5


title: RRBS_TSC1_WT_single_cell_sorted_cl5

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 5) 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 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: D13_IL2_REP6


title: Progenitor keratinocytes, bio 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. Biological replicates were performed in all cases using primary cells from at least 2 independent, unrelated donors.


title: D13_IL2_REP2


title: shDDX21-3 treated, differentiating keratinocytes, bio rep 2


title: D13_ZA_REP6


title: D13_IL2_REP1


title: RRBS_TSC1_WT_single_cell_sorted_cl4

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 4) 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 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.


title: D0_REP6


title: D0_REP5


title: D0_REP4


title: RRBS_TSC1_WT_single_cell_sorted_cl3

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 3) 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 3) 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: D13_ZA_REP2


title: RRBS_TSC1_WT_single_cell_sorted_cl30

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 30) 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 30) 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: D0_REP3

growth_protocol: PBMCs cultured at 5x10^6 cells/ml in RPMI-1640 + 10% FBS


title: D0_REP1


title: SN3595_2D_FBS_typI

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 mapped to the mouse transcriptome (UCSC annotation, genome build mm9) using rsem v1.3.1 and bowtie v1.2.3 with default options.
Assembly: mm9
Supplementary files format and content: Microsoft Excel file includes estimated read counts (from rsem) per gene for each sample

source_name: lung

growth_protocol: FNHCS media in 90% matrigel drops (3D) until treatment, all conditions maintained in a humidified 37ºC / 5% CO2 incubator at ambient O2 pressure on a 6 well plate (GenClone 25-105)

extract_protocol:
Qiagen RNeasy Kit
NEB Ultra II RNAseq

treatment_protocol: Cells were grown in indicated treatment for 7 days prior to dissociation with TrypLE and processing

value: lung

value: Alveolar organoid

value: Sftpc-eGFP

value: 2D in Ad-DMEM F/12 with 10% FBS


title: D13_IL2_REP3


title: shDDX21-3 treated, differentiating keratinocytes, bio rep 1


title: RRBS_TSC1_WT_single_cell_sorted_cl2

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 2) 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 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: D0_REP2


title: shDDX21-2 treated, differentiating keratinocytes, bio rep 1


title: SN3595_2D_FNH_AT2_AT1

value: 2D with FNHCS media


title: SN3593_2D_FBS_typI


title: RRBS_TSC1_WT_single_cell_sorted_cl29

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 29) 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 29) 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: Fyco1KO-P0C

city: Baltimore

first: S. Amer

last: Riazuddin

address: 600 N. Wolfe street

institute: Johns Hopkins University

data_processing:
Illumina Casava1.8 used for basecalling and de-indexing.
The paired-end reads were aligned to the Mus musculus genome (GRCm38/mm10) using the STAR algorithm (Ver. 2.5).
The HTSeq (Ver. 0.6.1) was used for the quantification of gene expression using mapped reads.
The expression data were normalized by calculating the fragment per kilobase per million mapped reads (FPKM) values for each gene (Mortazavi et al., Nat Methods, 2008)
Differential gene expression analysis was performed using the DESeq2 (Ver. 2_1.6.3).
Genome_build: mm10
Supplementary_files_format_and_content: Tab-delimited text file include FPKM values for each Sample

source_name: Ocular lens

extract_protocol:
The wild-type (Wt) and Fyco1-/- mice at P0 were first anesthetized with isoflurane and subsequently euthanized through cervical dislocation, and the lenses were isolated using forceps under a microscope. Briefly, three biological replicates, each consisting of ten lenses for Wt and Fyco1-/- mice were used for RNA-Seq library preparation. Upon extraction, lenses were dissolved immediately in Trizol reagent and total RNA was extracted for RNA-Seq library prepration.
NEB Next Ultra RNA Library Prep Kit was used to prepare RNA-Seq libraries using total RNA isolated from Wt and Fyco1-/- mouse lenses at P0.

tag: developmental stage

value: Postnatal day 0

value: Ocular lens


title: Fyco1KO-P0B


title: RRBS_TSC1_WT_single_cell_sorted_cl28

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 28) 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 28) 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: shControl treated, differentiating keratinocytes, bio rep 1


title: SN3593_2D_FNH_AT2_AT1

tag: cell type


title: RRBS_TSC1_WT_single_cell_sorted_cl27

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 27) 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 27) 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


title: Fyco1KO-P0A


title: SN3593_3D_FNH_typII

value: control, 3D with FNHCS media


title: SN3595_3D_FNH_typII


title: RRBS_TSC1_WT_single_cell_sorted_cl26

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 26) 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 26) 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: Progenitor, HA epitope, donor 2, input control

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.

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.

value: Progenitor


title: SN2294_GFP_minus

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

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

value: SN2294_GFP-, eGFP- lung cells from Sftpc-eGFP transgenic mice

value: FACS sorted for eGFP-


title: shDDX21-2 treated, differentiating keratinocytes, bio rep 2

value: DDX21 shRNA-3


title: WT-P0C

value: WT


title: Progenitor, HA epitope, donor 1, input control


title: SN2293_GFP_minus

value: SN2293_GFP-, eGFP- lung cells from Sftpc-eGFP transgenic mice


title: Progenitor, HA epitope, donor 2, ChIP-seq


title: RRBS_TSC1_WT_single_cell_sorted_cl24

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 24) 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 24) 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_TSC1_WT_single_cell_sorted_cl21

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 21) 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 21) 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: SN2285_GFP_minus

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


title: HAP1 DEGS1 KO, untreated, rep3

city: Aachen

first: Lars

last: Buschmann

address: Pauwelsstraße 30

department: Human Genetics

institute: RWTH University Hospital Aachen

data_processing:
Raw data files were demultiplexed, and FASTQ files were generated with bcl2fastq (version 2.20)
Data was aligned with BioJupies (Version 1.1.4)
Assembly: hg19
Supplementary files format and content: RNAseq_HAP1_DEGS1-rawdata.txt (Tab-delimited file containing raw counts for each sample)

growth_protocol: HAP1 cells were grown in IMDM (Gibco) supplemented with 5% FBS (Gibco) and P/S (Gibco), passaged 3 times a week, grown 24 hrs after passage before RNA isolation

extract_protocol:
Total RNA was extracted using Trizol (VWR), with following precipitation using Isopropanol and 2 70% ethanol washes, each time at 12000 x g. RNA pellet was dissolved in nuclease-free water (Qiagen) and stored at -70°C before library preparation.
1 µg of total RNA was used for library preparation using the Illumina TruSeq Stranded Total RNA kit following the manufacturers protocol.
high-output 1 × 75 flow cell

value: DEGS1 knockout

value: untreated


title: Progenitor, DDX21 epitope, donor 2, input control


title: SN2286_GFP_minus

value: Alveolar organoid

value: SN2286_GFP-, eGFP- lung cells from Sftpc-eGFP transgenic mice


title: HAP1 DEGS1 KO, untreated, rep2


title: HAP1 DEGS1 KO, untreated, rep1


title: WT-P0B


title: SN2294_GFP_plus

value: SN2294_GFP+, eGFP+ lung cells from Sftpc-eGFP transgenic mice

value: FACS sorted for eGFP+


title: RRBS_TSC1_WT_single_cell_sorted_cl23

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 23) 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 23) 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: Progenitor, HA epitope, donor 1, ChIP-seq


title: Progenitor, DDX21 epitope, donor 2, ChIP-seq


title: RRBS_TSC1_WT_single_cell_sorted_cl25

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 25) 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 25) 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: Progenitor, DDX21 epitope, donor 1, input control

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.


title: Progenitor, DDX21 epitope, donor 1, ChIP-seq


title: HAP1 WT, untreated, rep3

value: wildtype


title: RRBS_TSC1_WT_single_cell_sorted_cl20

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 20) 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 20) 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: SN2286_GFP_plus

value: SN2286_GFP+, eGFP+ lung cells from Sftpc-eGFP transgenic mice


title: HAP1 WT, untreated, rep2


title: WT-P0A

value: Postnatal day 0

value: Ocular lens


title: SN2293_GFP_plus

value: lung cells

value: SN2293_GFP+, eGFP+ lung cells from Sftpc-eGFP transgenic mice


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

value: Normal glucose


title: RRBS_TSC1_WT_single_cell_sorted_cl1

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 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: TSCs (TSC1 line, 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: RRBS_TSC1_WT_single_cell_sorted_cl17

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 17) 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 17) 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: HAP1 WT, untreated, rep1


title: RRBS_TSC1_WT_single_cell_sorted_cl16

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 16) 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 16) 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: Differentiating, normal glucose, HA epitope, donor 2, ChIP-seq


title: RRBS_TSC1_WT_single_cell_sorted_cl22

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 22) 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 22) 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: Differentiating, normal glucose, HA epitope, donor 1, input control


title: RRBS_TSC1_WT_single_cell_sorted_cl15

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 15) 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 15) 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: Differentiating, normal glucose, HA epitope, donor 1, ChIP-seq


title: ifos100_C

city: Gainesville

first: Christopher

last: Martyniuk

state: FL

address: 8891 SW 79th Avenue

institute: University of Florida

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

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.

value: larvae

value: 100 ug/L ifosfamide


title: PD67_PROCAP_B

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: ifos100_D


title: shControl treated, differentiating keratinocytes, bio rep 2

treatment_protocol: shRNAs targeting DDX21 were designed based on Sigma sequences (https://www.sigmaaldrich.com/US/en/semi-configurators/shrna?activeLink=productSearch) and were cloned into pLKO.1-puro or pLKO.1-blasti plasmid (Addgene, #10878 and #26655). The targeting sequences are: shScramble, 5’-TCCTAAGGTTAAGTCGCCCTCG-3’; shDDX21-2, 5’-GCATGAGGAATGGGATTGATA-3’; shDDX21-3, 5’-CCCATATCTGAAGAAACTATT-3’. For infection, the optimized viral titer was added to primary human keratinocytes together with polybrene (5 µg/mL). The next day, fresh media was added, and cells were selected using puromycin (1 µg/mL).

value: DDX21 shRNA-2


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


title: RRBS_TSC1_WT_single_cell_sorted_cl14

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 14) 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 14) 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: PD26_PROCAP_A

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

tag: cell line

value: proliferating

value: No


title: RRBS_TSC1_WT_single_cell_sorted_cl13

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 13) 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 13) 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: Differentiating, normal glucose, DDX21 epitope, donor 2, ChIP-seq

tag: treatment


title: RRBS_TSC1_WT_single_cell_sorted_cl12

description: Bisulfite-converted DNA

growth_protocol: TSCs (TSC1 line, clone 12) 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 12) 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: PD26_PROCAP_B


title: ifos100_A