title: K562_Harr_rep2

description:
Ribo-Seq/small RNA
RPF
processed data file: All_cell_lines_novel_coding_smORFs.txt

data_processing:
Library strategy: Ribo-seq
Raw sequencing data was demultiplexed and converted into FASTQ files using CASAVA (v1.8.2).
For Ribo-Seq short fragment RNA samples, reads were first trimmed of 3' adaptor sequence (AGATCGGAAGAGCACACGTCT) using fastx_clipper version 0.0.13 with the parameters: fastx_clipper -Q33 -l 20 -n. Trimmed reads were then aligned to a contaminant database of rRNA and tRNA sequences using Star version 2.5.2b with the parameters: --outSAMstrandField intronMotif --outReadsUnmapped Fastx --genomeDir /genomes/hg19cont.star --runThreadN 16. Unaligned reads were then aligned to hg19 with refGene annotations using the parameters: --outFilterMismatchNmax 2 --outFilterMultimapNmax 4 --chimScoreSeparation 10 --chimScoreMin 20 --chimSegmentMin 15 --outSAMattributes All --outSAMstrandField intronMotif. Secondary alignments were filtered out with samtools version 1.3 using the parameters: view -b -F 0X100. RibORF was used to score translation of smORFs. smORFs with RibORF scores of 0.7 and greater were considered translated. Any smORFs found to overlap with UCSC annotated CDS regions using bedtools version 2.25 or had BLASTP scores of 40 or greater against the Human RefSeq database were filtered out.
For RNA-Seq, reads were aligned to the hg19 using the STAR version 2.5.1b using the parameters: --outFilterIntronMotifs RemoveNoncanonical. Cufflinks was used for de novo transcriptome assembly of RNA-Seq data using parameters --multi-read-correct --frag-bias-correct and --library-type fr-firststrand options. Other parameters were set to default values. The resulting transcriptome was then 3-frame translated into ORFs using custom software.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: All_cell_lines_novel_coding_smORFs.txt: Tab-delimited text file containing the coordinates for all identified novel protein-coding smORFs, including microprotein sequence, exons, start site type, number of exons, cell lines detected in, samples detected in, average RPKM for each cell line, and PhyloCSF score for each exon.
Supplementary_files_format_and_content: Transcripts assembled by Cufflinks for HeLa-S3 and K562 in gtf format.

source_name: K562 (Human Chronic Myelogenous Leukemia)

extract_protocol:
For all ribosome footprinting experiments, adherent cells were grown to about 80% confluency in 10 cm or 15 cm diameter tissue culture dishes and suspension cells were grown to a density of approximately 500,000 cells/mL. Cells were washed with 5 mL ice-cold Phosphate Buffered Saline (PBS) with 100 µg/mL cycloheximide (CHX) added. Immediately after removing PBS, 400 or 800 µL of ice-cold lysis buffer (20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, with 1 mM DTT, 25 U/mL Turbo DNase (Thermo Fisher, AM2238), and 100 µg/mL CHX added fresh) was dripped onto the plate or added to the cell pellet. Cells were incubated on ice in lysis buffer for 10 min with periodic vortexing and pipetting to disperse the cells. The lysate was then clarified by centrifugation at 15,000 g for 10 min. Cell lysates were flash frozen and liquid nitrogen and stored at -80ºC for up to 5 d prior to ribosome footprinting. For experiments profiling translation initiation, the same procedure was followed except for the addition of either 2 µg/mL harringtonine (abcam) for 2 min or 20 µg/mL lactimidomycin (Calbiochem) for 30 min to media prior to PBS wash and lysis. A variety of digestion conditions were tested in this study. Briefly, RNA digestions using 250 U RNase I (Thermo Fisher, AM2294) per 100 µL lysate were used in the low resolution 293T and HeLaS3 experiments. For high-resolution experiments, 15 to 30 U TruSeq Nuclease (Illumina) was used to digest 30 to 60 µg RNA in up to 300 µL lysate. Digestion reactions were run for 45 to 60 min at RT and quenched with 100 to 200 U Superase-In RNase I inhibitor (Thermo Fisher) on ice. Following digestion, ribosome protected fragments (RPF) were purified from small RNA fragments using MicroSpin S-400 HR columns (GE Life Sciences) according to the TruSeq Ribo Profile Kit (Illumina). Low resolution experiments were cleaned up with Zymo RNA Clean & Concentrator-25 kit, while high resolution experiments were purified by acid phenol:chloroform extraction followed by isopropanol precipitation. Ribosomal RNAs were depleted from RPF fragments by Ribo-Zero Mammalian Kit (Illumina) following the manufacturer’s protocol.
Ribo-Seq and short fragment RNA-Seq libraries were prepared utilizing the Illumina TruSeq Ribo Profile kit according to the manufacturer's instructions.

treatment_protocol: For translation initiation experiments, cells were treated with 2µg/mL harringtonine (Harr) for 2 min at 37ºC or 20 µg/mL lactimidomycin (LTM) for 30 min at 37ºC.

value: Human Chronic Myelogenous Leukemia

value: Harringtonine


title: 293T_TotRNA_DMSO_rep2

status: Public on Jul 03 2019

type: SRA

city: La Jolla

first: Thomas

middle: Farid

last: Martinez

state: CA

address: 10010 N Torrey Pines Rf

department: Peptide Biology Laboratories

country: USA

institute: Salk Institute for Biological Studies

description: processed data file: All_cell_lines_novel_coding_smORFs.txt

library_source: transcriptomic

data_processing:
Raw sequencing data was demultiplexed and converted into FASTQ files using CASAVA (v1.8.2).
For Ribo-Seq short fragment RNA samples, reads were first trimmed of 3' adaptor sequence (AGATCGGAAGAGCACACGTCT) using fastx_clipper version 0.0.13 with the parameters: fastx_clipper -Q33 -l 20 -n. Trimmed reads were then aligned to a contaminant database of rRNA and tRNA sequences using Star version 2.5.2b with the parameters: --outSAMstrandField intronMotif --outReadsUnmapped Fastx --genomeDir /genomes/hg19cont.star --runThreadN 16. Unaligned reads were then aligned to hg19 with refGene annotations using the parameters: --outFilterMismatchNmax 2 --outFilterMultimapNmax 4 --chimScoreSeparation 10 --chimScoreMin 20 --chimSegmentMin 15 --outSAMattributes All --outSAMstrandField intronMotif. Secondary alignments were filtered out with samtools version 1.3 using the parameters: view -b -F 0X100. RibORF was used to score translation of smORFs. smORFs with RibORF scores of 0.7 and greater were considered translated. Any smORFs found to overlap with UCSC annotated CDS regions using bedtools version 2.25 or had BLASTP scores of 40 or greater against the Human RefSeq database were filtered out.
For RNA-Seq, reads were aligned to the hg19 using the STAR version 2.5.1b using the parameters: --outFilterIntronMotifs RemoveNoncanonical. Cufflinks was used for de novo transcriptome assembly of RNA-Seq data using parameters --multi-read-correct --frag-bias-correct and --library-type fr-firststrand options. Other parameters were set to default values. The resulting transcriptome was then 3-frame translated into ORFs using custom software.
Genome_build: hg19 (GRCh37)
Supplementary_files_format_and_content: All_cell_lines_novel_coding_smORFs.txt: Tab-delimited text file containing the coordinates for all identified novel protein-coding smORFs, including microprotein sequence, exons, start site type, number of exons, cell lines detected in, samples detected in, average RPKM for each cell line, and PhyloCSF score for each exon.
Supplementary_files_format_and_content: Transcripts assembled by Cufflinks for HeLa-S3 and K562 in gtf format.

molecule: total RNA

organism: Homo sapiens

source_name: HEK293T (Human Embryonic Kidney)

growth_protocol: All cell lines were maintained at 37ºC with 5% CO2. HEK293T and HeLa-S3 cells were grown in DMEM supplemented with 10% FBS. K562 cells were grown in RPMI 1640 supplemented with 10% FBS.

extract_protocol:
For accompanying short fragment total RNA-Seq experiments, 50 µL of cell lysate from cells prepped for Ribo-Seq was removed and RNA was extracted by acid phenol:chloroform extract followed by isopropanol precipitation. Total RNA was then heat fragmented at 95ºC for 25 min prior to library construction.
Ribo-Seq and short fragment RNA-Seq libraries were prepared utilizing the Illumina TruSeq Ribo Profile kit according to the manufacturer's instructions.

treatment_protocol: For ER stress experiments, cells were treated with either 0.1% DMSO, 5µg/mL tunicamycin (TM), or 1µM thapsigargin (TG) for 4 h at 37ºC.

tag: cell line

tag: cell type

value: Human Embryonic Kidney

tag: treatment

value: DMSO


title: 293T_TotRNA_DMSO_rep1


title: HeLa-S3_LTM_rep2

source_name: HeLa-S3 (Human Cervix Adenocarcinoma)

value: Human Cervix Adenocarcinoma

value: Lactimidomycin


title: K562_Harr_rep1


title: 293T_TotRNA_TM_rep1

value: Tunicamycin


title: HeLa-S3_Harr_rep2


title: HeLa-S3_Harr_rep1


title: 293T_LTM_rep2


title: 293T_LTM_rep1


title: K562_Ribo_HiRes_rep2


title: K562_Ribo_HiRes_rep1


title: K562_Ribo_HiRes_rep3


title: HeLa-S3_LTM_rep1


title: 293T_Harr_rep1


title: input DNA 30 min

city: Boston

first: Yuko

last: Hasegawa

state: MA

address: 240 Longwood Ave

department: BCMP

institute: Harvard Medical School

library_source: genomic

data_processing:
ChIP-seq reads were aligned to the custom genome containing hg38 and sacCer3 assembly using bowtie2 using parameter --sensitive.
PCR duplicates were removed using picard MarkDuplicates and mapped reads with low quality score (less than 30) were excluded using samtools.
Peaks calling was performed with MACS2 using input from each expetiment as a reference with the following setting: -q 0.01.
bedGraph files were created by MACS2 pileup.
Values in bedGraph were normalized to reference-adjusted reads per million (RRPM) calculated as deviding human ChIP-seq score by total yeast mapped reads.
bedGraph files were converted to bigWig by UCSC bedGraphToBigWig program.
Genome_build: hg38
Supplementary_files_format_and_content: bigWig, Scores represent Reference-adjusted RPM

molecule: genomic DNA

growth_protocol: pCMV-TBP-ERT2-3HA construct was transfected to HEK293 (ATCC) cells, and the stable line was established by G418 selection. Cells were routinely cultured in phenol-red free DMEM containing 10% FBS.

extract_protocol:
Cell Lysates were clarified from sonicated nuclei and ERT2-3HA fused TBP-DNA complexes were isolated with HA antibody.
Libraries were prepared according to NEB's instructions accompanying the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645).

treatment_protocol: Cells were treated with EtOH or 100nM 4OHT.

tag: chip antibody


title: input DNA 60 min


title: HeLa-S3_Ribo_HiRes_rep2


title: input DNA 1440 min


title: TBP-ERT2-3HA ChIP-seq EtOH


title: input DNA 15 min


title: HeLa-S3_Ribo_LoRes_rep2


title: K562_TotalRNA

description: processed data file: K562_Cufflinks_transcript_assembly.gtf

extract_protocol:
For paired-end sequencing, total RNA for RNA-Seq was prepared using the Qiagen RNeasy kit with gDNA elimination columns according to the manufacturer's instructions.
RNA-Seq libraries were prepared utilizing both the Illumina TruSeq Stranded mRNA kit and the Illumina TruSeq Stranded Total RNA kit according to manufacturer instructions.


title: HeLa-S3_Ribo_HiRes_rep1


title: HeLa-S3_Ribo_LoRes_rep1


title: 293T_Ribo_TG_rep2

value: Thapsigargin


title: 293T_Ribo_TG_rep1


title: 293T_Ribo_TM_rep2


title: 293T_Ribo_TM_rep1


title: input DNA 360 min


title: 293T_Ribo_DMSO_rep2


title: 293T_Ribo_DMSO_rep1


title: KDM2AB_CXXCfl_TAM_nativeinput_rep3

city: Oxford

first: Anne

last: Turberfield

address: South Parks Road

department: Biochemistry

country: United Kingdom

institute: University of Oxford

data_processing:
For RNAPII, KDM2A and KDM2B cChIP-seq reads were aligned to concatenated mm10 and hg19 genomes and for histone ChIP-seq reads were aligned to the mouse mm10 genome, using Bowtie 2 with the ‘-- no-mixed’ and ‘-- no-discordant’ options (Langmead and Salzberg 2012). Reads that were mapped more than once were discarded, and PCR duplicates were removed using SAMTools (Li et al. 2009).
To internally calibrate RNAPII, KDM2A and KDM2B ChIP-seq, we spiked a fixed number of HEK293T cells into each sample. For data visualisation and annotation of genomic intervals, mm10 reads were randomly subsampled by a factor that reflects the total number of spike-in reads in the same sample, as previously described (Bonhoure et al. 2014; Orlando et al. 2014; Hu et al. 2015). To account for any variation in the exact spike-in cell: mESC mixing ratio between biological replicates, the subsampling factors were additionally corrected according to the ratio of hg19/mm10 total read counts in the matched input sample.
For each condition, biological replicates were merged for downstream applications. Genome coverage tracks were generated using the pileup function from MACS2 (Zhang et al. 2008). Bigwig tracks of H3K36me2 normalised to H3 were generated from merged bigwig files using the deepTools bigwigCompare function with ‘--operation ratio’ setting (Ramírez et al. 2016).
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files representing genome coverage for merged replicates.

organism: Mus musculus

source_name: Mouse embryonic stem cells, tamoxifen treated, gDNA-seq

growth_protocol: Mouse embryonic stem cells were cultured on gelatine-coated dishes at 37oC and 5% CO2 in Dulbecco’s Modified Eagle Medium (Life Technologies) supplemented with 15% fetal bovine serum (Labtech), 2mM L-glutamine (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), 1x non-essential amino acids (Life Technologies), 1x penicillin-streptomycin (Life Technologies), and 10 ng/ml leukemia-inhibitory factor. Human HEK293T cells were grown at 37oC and 5% CO2 in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 0.5 mM beta-mercaptoethanol and 1x penicillin-streptomycin.

extract_protocol: For histone H3 and H3K36me2 ChIP, 1x107 mESCs were crosslinked for 10 min in 1% formaldehyde. Reactions were quenched by addition of 125 mM glycine. The released nuclei were washed twice in PBS, then resuspended in lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1 and 1xPIC) and incubated on ice for 30 min. For KDM2A/B ChIP, 5x107 mESCs were resuspended in PBS and mixed with 2x106 HEK293T cells. Cells were crosslinked in 2 mM disuccinimidyl glutarate (Thermo Scientific) for 45 min at 25°C with gentle rotation, then in 1% formaldehyde for 12.5 min (methanol-free, Life Technologies). Reactions were quenched by addition of 125 mM glycine, and crosslinked cells were resuspended in lysis buffer (50mM HEPES-KOH pH 7.9, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% TritonX-100 and 1x PIC) and rotated for 10 min at 4°C. The released nuclei were washed (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA and 1x PIC) for 5 min at 4°C, and the nuclear pellet resuspended in 1 ml sonication buffer (10mM Tris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine and 1xPIC). For RNAPII ChIP, 5x107 mESCs were resuspended in PBS and mixed with 4x106 HEK293T cells. Cells were crosslinked for 10 min in 1% formaldehyde. Reactions were quenched by addition of 150 mM glycine, and the crosslinked cells resuspended in FA-lysis buffer for 10 min (50mM HEPES pH 7.9, 150mM NaCl, 2mM EDTA, 0.5mM EGTA, 0.5% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 10mM NaF, 1mM AEBSF, 1xPIC(Roche)).  For H3, H3K36me2, KDM2A/B or RNAPII ChIP, chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to approximately 0.5 kb. Sonicated chromatin was diluted 10-fold in ChIP dilution buffer (1% TritonX-100, 1mM EDTA, 20mM TrisHCl pH 8, 150mM NaCl and 1x PIC) for H3, H3K36me2 or KDM2A/B ChIP, or in FA-lysis buffer for RNAPII ChIP. Chromatin was pre-cleared for 1 hour with either protein A agarose beads (Repligen, for H3, H3K36me2 or RNAPII ChIP) or protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA. For each ChIP reaction, 150 μg chromatin (KDM2A/B), 300 μg chromatin (RNAPII) or chromatin corresponding to 1x105 cells (H3 or H3K36me2 ChIP) was incubated overnight with the appropriate antibody: anti-H3 (in house, 15 μl), anti-H3K36me2 (in house, 15 μl), anti-KDM2A (in house, 2.4 μl), anti-KDM2B (in house, 2 μl), anti-Rbp1-NTD (CST D8L4Y, 15 μl), anti-Rbp1-CTD-Ser5P (CST D9N5I, 12.5 μl), anti-Rbp1-CTD-Ser2P (CST E1Z3G, 12.5 μl). Antibody-bound chromatin was isolated using blocked protein A agarose (H3, H3K63me2 or RNAPII ChIP) or magnetic beads (KDM2A/B ChIP) for 2 hours at 4°C. For H3, H3K36me2 or KDM2A/B ChIP, washes were performed with low salt buffer (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris-HCl pH 8, 150mM NaCl), high salt buffer (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM TrisHCl pH 8, 500mM NaCl), LiCl buffer (250mM LiCl, 1% NP40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCl pH 8) and two washes with TE buffer (10mM Tris-HCl pH 8, 1mM EDTA). For RNAPII ChIP, washes were performed with FA-Lysis buffer, FA-Lysis buffer containing 500mM NaCl, DOC buffer (250mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 2mM EDTA, 10mM Tris-HCl pH 8) and two washes with TE buffer. ChIP DNA was eluted in elution buffer (1% SDS, 100mM NaHCO3) and crosslinks reversed overnight at 65°C with 200mM NaCl and 2 μl RNase A (Sigma). A matched input sample (corresponding to 10% of original ChIP reaction) was treated identically. The following day, samples were treated with 20 μg/ml Proteinase K (Sigma) for 2 hours at 45°C and purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research). For H2AK119ub1 and H3K27me3 native ChIP-seq, 5 x 107 mouse ESCs were resuspended in 1ml RSB (10mM Tris-HCl pH 8.0, 10mM NaCl, 3mM MgCl2, 5mM N-ethylmaleimide (NEM)), then lysed by addition of 28 ml RSB containing 0.1% NP40. The released nuclei were washed in RSC buffer (RSB supplemented with 0.25M sucrose and 3mM CaCl2), then resuspended in 1 ml RSC buffer containing  10mM NEM and 1xPIC(Roche). Each sample was then incubated with 150 units of MNase (Fermentas) at 37°C for 5 min, then 4 mM EDTA was added to halt MNase digestion. Following centrifugation at 5000 rpm for 5 min at 4°C, the supernatant (S1) was retained. The pellet was resuspended in 300 µl nucleosome release buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 0.2mM EDTA, 10mM NEM, 1xPIC), incubated at 4°C for 1 h, then passed five times through a 27G needle using a 1 ml syringe. After centrifugation at 5000 rpm for 5 min at 4°C, the second supernatant (S2) was combined with S1. A small aliquot of S1/S2 DNA was purified and visualized on a 1.5% agarose gel to confirm digestion to predominantly mono-nucleosomes. S1/S2 was diluted 10-fold in native ChIP incubation buffer (70mM NaCl, 10mM Tris-HCl pH 7.5, 2mM MgCl2, 2mM EDTA, 0.1% TritonX-100, 10mM NEM, 1xPIC). For each ChIP reaction, 1 ml of diluted nucleosomes was incubated overnight at 4°C with 5 μl of anti-H2AK119ub1 (CST D27C4) or anti-H3K27me3 (in-house). Antibody-bound nucleosomes were isolated by incubation for 1 hour at 4°C with protein A agarose beads, which were pre-blocked overnight in native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA. The beads were then washed four times with native wash buffer (20mM Tris-HCl pH 7.5, 2mM EDTA, 125mM NaCl, 0.1% TritonX-100) and once with TE buffer. ChIP DNA was eluted using 100 ul of elution buffer (1% SDS, 0.1 M NaHCO3), then purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research). DNA from a matched input sample (corresponding to 10% of original ChIP reaction) was also purified.

treatment_protocol: mESC lines were treated with 800 nM 4-hydroxytamoxifen (Sigma) for 96 hours to induce KDM2 long isoform knockout (Kdm2a/b-JmjCfl/fl) or ZF-CxxC domain deletion (Kdm2a/b-CXXCfl/fl).

tag: tissue

value: embryonic stem cells

tag: genotype

tag: replicate

value: tamoxifen

value: none (input)


title: slamseq_WT_0h

city: New York

first: Samie

last: Jaffrey

state: New York

address: 1300 York Ave

department: Pharmacology

institute: Weill Cornell Medicine

description:
4sU-incorporated with hours of U chase
slamseq.txt

data_processing:
Library strategy: SLAM-Seq
For miCLIP,  adapters and low-quality based were trimmed using flexbar v2.5: flexbar -n 20 -as AGATCGGAAGAGCGGTTCAG -f i1.8 --pre-trim-phred 30 -t output -r Read1.fastq -p Read2.fastq
Reads were then demultiplexed using pyBarcodeFilter.py and de-deduplicted using pyFastqDuplicateRemover.py from the pyCRAC software suite, and processed for the CTK iCLIP/miCLIP protocol. Second mate reads were reverse complimented and concatenated a with the first read file and aligned to GRCh38 (or mm10 for MEFs) using bwa v0.7.17: bwa aln -t 4 -n 0.06 -q 20 GRCh38.fasta ConcatendatedReads.fasta > AlignedReads.sai
Aligned reads were then processed as in miCLIP using C to T transitions to identify m6A sites. PCIF1 KO was used as a negative control for WT to determine regions in the genome which are dependent on PCIF1 and are thus m6Am. Ad hoc analyses were performed on the command line making extensive use of bedtools and deeptools.
Ribo-seq was trimmed and demultiplexed as with miCLIP. These reads and those for SLAM-seq were aligned first to the ribosomal DNA, and those that unmapped were mapped to the transcriptome using STAR aligner.
miCLIP Barcodes: WT: NNNCAAT; KO1: NNNGGTT; KO2: NNNTTGT; MEF1: NNNGGCG; MEF2: NNNCCGG
Genome_build: hg38/mm10
Supplementary_files_format_and_content: Processed data are in bed files: m6A.DRACH.FDR01.bed files contain called m6A sites with FDR<0.1; strict_putatitive_m6Am.bed contains the PCIF1-dependent bins; m6Am_locations.binsFINAL.bed contains the coordinates for the m6Am nucleotide in each region

molecule: polyA RNA

extract_protocol:
Total RNA was harvested using Trizol, and polyA mRNA was enriched using 2 rounds of Dynabead oligoDT selection.
Libraries were constructed using either NEBNext Ultra II Directional RNA library prep kit (inputs/SLAM-seq), or using the miCLIP cloning protocol (miCLIP/Ribo-seq)

value: WT

tag: hours of u chase


title: KDM2AB_CXXCfl_UNT_nativeinput_rep3

source_name: Mouse embryonic stem cells, untreated, gDNA-seq

value: untreated


title: KDM2AB_CXXCfl_TAM_nativeinput_rep1


title: slamseq_WT_12h


title: slamseq_WT_6h


title: KDM2AB_CXXCfl_TAM_nativeinput_rep2


title: 293T_Ribo_MedRes


title: 293T_Ribo_HiRes


title: 293T_Ribo_LoRes


title: KDM2AB_JmjCfl_TAM_nativeinput_rep1


title: KDM2AB_CXXCfl_UNT_nativeinput_rep2


title: slamseq_PCIF1KO_6h

value: PCIF1 KO


title: KDM2AB_JmjCfl_UNT_nativeinput_rep3


title: KDM2AB_CXXCfl_UNT_nativeinput_rep1


title: KDM2AB_JmjCfl_UNT_nativeinput_rep1


title: KDM2AB_JmjCfl_TAM_nativeinput_rep2


title: KDM2AB_JmjCfl_TAM_nativeinput_rep3


title: slamseq_PCIF1KO_0h


title: KDM2AB_JmjCfl_UNT_nativeinput_rep2


title: riboseq_WT_2

description:
Ribosome protected fragments
ribosome_protected_fragment.txt; translation_efficiency.txt

data_processing:
Library strategy: Ribo-seq
For miCLIP,  adapters and low-quality based were trimmed using flexbar v2.5: flexbar -n 20 -as AGATCGGAAGAGCGGTTCAG -f i1.8 --pre-trim-phred 30 -t output -r Read1.fastq -p Read2.fastq
Reads were then demultiplexed using pyBarcodeFilter.py and de-deduplicted using pyFastqDuplicateRemover.py from the pyCRAC software suite, and processed for the CTK iCLIP/miCLIP protocol. Second mate reads were reverse complimented and concatenated a with the first read file and aligned to GRCh38 (or mm10 for MEFs) using bwa v0.7.17: bwa aln -t 4 -n 0.06 -q 20 GRCh38.fasta ConcatendatedReads.fasta > AlignedReads.sai
Aligned reads were then processed as in miCLIP using C to T transitions to identify m6A sites. PCIF1 KO was used as a negative control for WT to determine regions in the genome which are dependent on PCIF1 and are thus m6Am. Ad hoc analyses were performed on the command line making extensive use of bedtools and deeptools.
Ribo-seq was trimmed and demultiplexed as with miCLIP. These reads and those for SLAM-seq were aligned first to the ribosomal DNA, and those that unmapped were mapped to the transcriptome using STAR aligner.
miCLIP Barcodes: WT: NNNCAAT; KO1: NNNGGTT; KO2: NNNTTGT; MEF1: NNNGGCG; MEF2: NNNCCGG
Genome_build: hg38/mm10
Supplementary_files_format_and_content: Processed data are in bed files: m6A.DRACH.FDR01.bed files contain called m6A sites with FDR<0.1; strict_putatitive_m6Am.bed contains the PCIF1-dependent bins; m6Am_locations.binsFINAL.bed contains the coordinates for the m6Am nucleotide in each region

molecule: cytoplasmic RNA


title: slamseq_PCIF1KO_12h


title: KDM2AB_CXXCfl_TAM_H3K27me3_rep2

source_name: Mouse embryonic stem cells, tamoxifen treated, H3K27me3 ChIP-seq


title: KDM2AB_CXXCfl_TAM_H3K27me3_rep3


title: KDM2AB_CXXCfl_TAM_H3K27me3_rep1


title: riboseq_PCIF1KO_3


title: riboseq_PCIF1KO_2


title: NS_SF.ATAC_MT_rep3

city: San Francisco

first: Nicole

last: Stone

address: 1650 Owens St.

institute: Gladstone Institutes - UCSF

description:
accessible genomic DNA
SF.ChIP.ATAC_featureCounts.txt

data_processing:
Reads were aligned to the mm9 genomic assembly using bowtie2 with options: -X 600 --no-mixed --no-discordant.
Duplicate reads were removed using Picard MarkDuplicates (http://broadinstitute.github.io/picard).
For each of the replicate samples, reads were assigned to regions of interest using featureCounts.
Counts were normalized for differences in sequencing depths for each replicate.
Genome_build: mm9
Supplementary_files_format_and_content: Text file containing normalized counts per region matrix

source_name: neonatal mouse heart

extract_protocol:
We prepared samples for ATAC-seq as previously described (Buenrostro et al. 2013). Aliquots of 50,000 cells were spun down (310 RCF for 3 minutes) and washed with 200 μL of chilled PBS. Samples were lysed with 200 μL of chilled lysis buffer (20 mM Tris-HCl (pH 8.0), 85 mM KCl, 0.5% NP-40) and spun down at 500 RCF for 5 minutes. Nuclear pellets were transposed with 25 μL of Tagment DNA Buffer, 2.5 μL of Tagment DNA Enzyme (Nextera Sample prep Kit from Illumina, cat #FC-121-1030), and 22.5 μL of nuclease-free H2O. The samples were incubated at 37°C for 30 minutes and stored at −20°C. Transposed samples were purified using the QIAGEN MinElute Reaction Cleanup Kit (cat #28204).
Libraries were amplified using 25 μL of NEBNext High Fidelity 2x PCR Master Mix, 1.25 μM Nextera custom primer, 1.25 μM Nextera custom primers with unique barcodes, and nuclease-free H2O. We amplified samples using the following PCR conditions: 72°C for 5 minutes; 98°C for 30 seconds; and cycled at 98°C for 10 seconds, 63°C for 30 seconds and 72°C for 1 minute. Half of each sample was amplified for 12 cycles, MinElute purified and assessed by bioanalyzer for library quality. Samples concentration was quantified by Qbit before pooling. Samples were sequenced at 100PE on the Illumina HiSeq 4000 at the UCSF CAT core.

treatment_protocol: Exogenous overexpression of reprogramming factors using retrovirus was performed as previously described (Ieda et al. 2010; Qian et al. 2013), on immortalized neonatal cardiac fibroblasts expressing large T antigen. Cells were infected with freshly prepared Gata4, Mef2c, and/or Tbx5 (GMT) retroviruses, and cultured with Tgfβ and Wnt inhibitors (sb431542 and xav939, respectively; Mohamed et al. 2017) prior to transposition of unsorted cell populations at day 2. We completed three biological replicates per condition.

tag: strain

value: alpha-MHC-GFP

value: neonatal cardiac fibroblast cell line expressing large T antigen

tag: exogenous retroviral expression

value: Mef2c, Tbx5

tag: chemical treatment

tag: reprogramming time point

value: day 2


title: KDM2AB_CXXCfl_UNT_H3K27me3_rep1

source_name: Mouse embryonic stem cells, untreated, H3K27me3 ChIP-seq


title: KDM2AB_CXXCfl_UNT_H3K27me3_rep3


title: KDM2AB_JmjCfl_TAM_H3K27me3_rep3


title: riboseq_input_WT_2

description:
Riboseq input
riboseq_input.txt


title: KDM2AB_CXXCfl_UNT_H3K27me3_rep2


title: riboseq_input_WT_1

description:
Riboseq input
riboseq_input.txt


title: KDM2AB_JmjCfl_TAM_H3K27me3_rep2


title: KDM2AB_JmjCfl_UNT_H3K27me3_rep3


title: KDM2AB_JmjCfl_TAM_H3K27me3_rep1


title: riboseq_PCIF1KO_1


title: riboseq_input_PCIF1KO_2

description:
Riboseq input
riboseq_input.txt


title: riboseq_input_PCIF1KO_3

description:
Riboseq input
riboseq_input.txt


title: KDM2AB_CXXCfl_TAM_H2AK119ub_rep3

source_name: Mouse embryonic stem cells, tamoxifen treated, H2AK119ub ChIP-seq


title: riboseq_WT_1


title: WGBS_PBL_E023

city: Darlinghurst

first: Stephen

last: Bradford

state: NSW

address: 384 Victoria St

country: Australia

institute: Garvan Institute of Medical Research

description:
E23BUF
Blood

data_processing:
Paired-end reads were adaptor and quality score trimmed using trim_galore v0.2.8. Trimmed reads were mapped to the hg19 genome build using bismark v0.8.3 {Krueger, 2011 #519} with the parameters “‐‐bowtie2 ‐X 1000“, technical replicates pooled using samtools v0.1.19 “merge” and PCR duplicates marked using Picard v1.91 “MarkDuplicates”. Methylation count data is extracted using bismark_methylation_extractor with the parameters “‐‐comprehensive ‐‐merge_non_CpG ‐‐bedgraph ‐‐counts ‐‐report ‐‐gzip ‐‐buffer_size 20G”.
Trimmed reads were mapped to the hg19 genome build using bismark v0.8.3 {Krueger, 2011 #519} with the parameters “‐‐bowtie2 ‐X 1000“, technical replicates pooled using samtools v0.1.19 “merge” and PCR duplicates marked using Picard v1.91 “MarkDuplicates”. Methylation count data is extracted using bismark_methylation_extractor with the parameters “‐‐comprehensive ‐‐merge_non_CpG ‐‐bedgraph ‐‐counts ‐‐report ‐‐gzip ‐‐buffer_size 20G”.
Methylation count data is extracted using bismark_methylation_extractor with the parameters “‐‐comprehensive ‐‐merge_non_CpG ‐‐bedgraph ‐‐counts ‐‐report ‐‐gzip ‐‐buffer_size 20G”.
Genome_build: hg19

source_name: PBL

extract_protocol:
SA and VA samples - In brief, freshly isolated VAT and SAT samples were weighed and minced with surgical scissors. For each gram of minced tissue, 1.25 mg/mL of Collagenase I (Sigma Aldrich, CAT#C6885-1G) was added to 2 mL of HEPES buffer containing 7.9 g/L NaCl, 323 mg/L CaCl2.2H2O, 308 mg/L MgSO4.7H2O, 154.3 mg/L Na2HPO4 (anhydrous), 5 mL/L of 1M Hepes (Sigma Aldrich), 450 mg/L D-glucose, pH 7.2 – 7.4, 20 g/L BSA. The tissue was incubated for 30-45 minutes at 37ºC before being diluted 1:9 with additional HEPES buffer. The mixture was passed through a 250 micron mesh before being centrifuged at 300 rcf for 6 minutes at room temperature. The top lipid layer was removed and the adipocytes collected for snap-freezing in liquid nitrogen.; VATsamples - Snap frozen in liquid Nirtogen; PBL - 10 mL blood was collected into K2EDTA vacutainers. Following centrifugation at 1,300 rcf for 10 minutes at room temperature, the top plasma layer was removed and the buffy coat layer collected. Cells were washed three times, with vigorous resuspension, in 10 mL Tris-EDTA buffer (100 mM Tris-HCl, 0.1 mM EDTA), with pellets collected after centrifugation at 10,000 rcf for 10 minutes. The final pellet was resuspended in 500 µL of buffer and stored at -80ºC. DNA was extracted: SAVA - using proteinase K digestion and phenol chloroform extraction; VAT - DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Cat#69504); PBL - DNA was extracted from PBL sample types following the Gentra Puregene Blood Cell Kit (Qiagen, Cat#158445).
Whole genome bisulfite sequencing libraries were prepared following Illumina’s “Whole- Genome Bisulfite sequencing for Methylation Analysis” protocol.  Briefly, 1 mg of genomic DNA was spiked with 0.5% unmethylated lambda DNA and sonicated to generate fragments of size between 150 to 300bp. Library preparation was performed for core samples using the Illumina’s Paired-end DNA Sample Prep Kit (discontinued, Illumina, CA, USA) according to the manufacturer’s protocol. The size selected libraries were then subjected to bisulfite conversion as previously described {Clark, 2006 #385}. Adaptor-ligated bisulfite treated DNA was enriched by 10 cycles of PCR amplification using the PfuTurbo Cx Hotstart DNA Polymerase (Stratagene). Qualitative and quantitative checks of the libraries were performed using Agilent’s High sensitivity DNA kit (Agilent) and KAPA Library quantification kit (KAPA Biosystems).

tag: gender

value: Female

tag: tissue origin

value: Blood


title: KDM2AB_CXXCfl_TAM_H2AK119ub_rep1


title: KDM2AB_JmjCfl_UNT_H3K27me3_rep1


title: riboseq_input_PCIF1KO_1

description:
Riboseq input
riboseq_input.txt


title: WGBS_PBL_E018

description:
E18BUF
Blood


title: miCLIP_PCIF1KO_1

description: miCLIP

data_processing:
Library strategy: miCLIP
For miCLIP,  adapters and low-quality based were trimmed using flexbar v2.5: flexbar -n 20 -as AGATCGGAAGAGCGGTTCAG -f i1.8 --pre-trim-phred 30 -t output -r Read1.fastq -p Read2.fastq
Reads were then demultiplexed using pyBarcodeFilter.py and de-deduplicted using pyFastqDuplicateRemover.py from the pyCRAC software suite, and processed for the CTK iCLIP/miCLIP protocol. Second mate reads were reverse complimented and concatenated a with the first read file and aligned to GRCh38 (or mm10 for MEFs) using bwa v0.7.17: bwa aln -t 4 -n 0.06 -q 20 GRCh38.fasta ConcatendatedReads.fasta > AlignedReads.sai
Aligned reads were then processed as in miCLIP using C to T transitions to identify m6A sites. PCIF1 KO was used as a negative control for WT to determine regions in the genome which are dependent on PCIF1 and are thus m6Am. Ad hoc analyses were performed on the command line making extensive use of bedtools and deeptools.
Ribo-seq was trimmed and demultiplexed as with miCLIP. These reads and those for SLAM-seq were aligned first to the ribosomal DNA, and those that unmapped were mapped to the transcriptome using STAR aligner.
miCLIP Barcodes: WT: NNNCAAT; KO1: NNNGGTT; KO2: NNNTTGT; MEF1: NNNGGCG; MEF2: NNNCCGG
Genome_build: hg38/mm10
Supplementary_files_format_and_content: Processed data are in bed files: m6A.DRACH.FDR01.bed files contain called m6A sites with FDR<0.1; strict_putatitive_m6Am.bed contains the PCIF1-dependent bins; m6Am_locations.binsFINAL.bed contains the coordinates for the m6Am nucleotide in each region

tag: antibody


title: miCLIP_WT

description:
miCLIP
strict_putative_m6Am.bed

value: Wild type


title: KDM2AB_CXXCfl_UNT_H2AK119ub_rep2

source_name: Mouse embryonic stem cells, untreated, H2AK119ub ChIP-seq


title: WGBS_VAT_E023

description:
E23VAT
Visceral Adipose

source_name: VAT

value: Visceral Adipose


title: KDM2AB_CXXCfl_TAM_H2AK119ub_rep2


title: KDM2AB_CXXCfl_UNT_H2AK119ub_rep3


title: WGBS_PBL_E013

description:
E13BUF
Blood


title: KDM2AB_CXXCfl_UNT_H2AK119ub_rep1


title: WGBS_VAT_E018

description:
E18VAT
Visceral Adipose


title: WGBS_VAT_E013

description:
E13VAT
Visceral Adipose


title: KDM2AB_JmjCfl_UNT_H3K27me3_rep2


title: KDM2AB_JmjCfl_TAM_H2AK119ub_rep2


title: miCLIP_PCIF1KO_2

description: miCLIP


title: WGBS_VA_E023

description:
E23VA
Visceral Adipocyte

source_name: VA


title: KDM2AB_JmjCfl_TAM_H2AK119ub_rep3


title: KDM2AB_JmjCfl_TAM_H2AK119ub_rep1


title: WGBS_SA_E018

description:
E18SA
Subcutaneous Adipocyte

source_name: SA

value: Subcutaneous Adipose


title: WGBS_VA_E013

description:
E13VA
Visceral Adipocyte


title: WGBS_SA_E023

description:
E23SA
Subcutaneous Adipocyte


title: RR_RNA_VAT_E023

description: Visceral Adipose

data_processing:
Adapters from the sequencing reads were trimmed using Trimmomatic
Reads were then mapped to the human genome (hg19) using custom bioinformatics pipeline NGSANE {Buske, 2014 #342}, which utilizes TopHat {Trapnell, 2013 #465} (TopHat v2.0.6) to perform the alignment in a strand specific manner.
Following the mapping process, htseqcount feature from the bioinformatics tool htseq {Anders, 2015 #466} was employed to count the number of uniquely mapped reads of genomic features such as genes and transcripts guided by the GENCODE GTF (v19) {Harrow, 2012 #468} annotation with the following parameter (HTSEQCOUNT_UNIQUE = 1 and GTF = “gencode.v19.annotation.gtf”)
Genome_build: hg19
Supplementary_files_format_and_content: count

extract_protocol:
SA and VA samples - In brief, freshly isolated VAT and SAT samples were weighed and minced with surgical scissors. For each gram of minced tissue, 1.25 mg/mL of Collagenase I (Sigma Aldrich, CAT#C6885-1G) was added to 2 mL of HEPES buffer containing 7.9 g/L NaCl, 323 mg/L CaCl2.2H2O, 308 mg/L MgSO4.7H2O, 154.3 mg/L Na2HPO4 (anhydrous), 5 mL/L of 1M Hepes (Sigma Aldrich), 450 mg/L D-glucose, pH 7.2 – 7.4, 20 g/L BSA. The tissue was incubated for 30-45 minutes at 37ºC before being diluted 1:9 with additional HEPES buffer. The mixture was passed through a 250 micron mesh before being centrifuged at 300 rcf for 6 minutes at room temperature. The top lipid layer was removed and the adipocytes collected for snap-freezing in liquid nitrogen.; VATsamples - Snap frozen in liquid Nirtogen; PBL - 10 mL blood was collected into K2EDTA vacutainers. Following centrifugation at 1,300 rcf for 10 minutes at room temperature, the top plasma layer was removed and the buffy coat layer collected. Cells were washed three times, with vigorous resuspension, in 10 mL Tris-EDTA buffer (100 mM Tris-HCl, 0.1 mM EDTA), with pellets collected after centrifugation at 10,000 rcf for 10 minutes. The final pellet was resuspended in 500 µL of buffer and stored at -80ºC. DNA was extracted: SAVA - using proteinase K digestion and phenol chloroform extraction; VAT - DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Cat#69504); PBL - DNA was extracted from PBL sample types following the Gentra Puregene Blood Cell Kit (Qiagen, Cat#158445).
RNA-seq libraries were prepared using Illumina TruSeq Stranded Total RNA Library Prep Kit 500 ng of RNA was used for each sample and ribosomal RNA reduction was done using


title: WGBS_SA_E013

description:
E13SA
Subcutaneous Adipocyte


title: RR_RNA_VAT_E018

description: Visceral Adipose


title: RR_RNA_VAT_E013

description: Visceral Adipose


title: KDM2AB_CXXCfl_TAM_KDM2AB-ChIP-input_rep2

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, tamoxifen treated, gDNA-seq

tag: spike in cells

value: human HEK293T cells


title: KDM2AB_CXXCfl_TAM_KDM2AB-ChIP-input_rep1


title: RR_RNA_VA_E023

description: Visceral Adipocyte


title: KDM2AB_JmjCfl_UNT_H2AK119ub_rep1


title: KDM2AB_CXXCfl_UNT_KDM2AB-ChIP-input_rep1

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, untreated, gDNA-seq


title: KDM2AB_CXXCfl_UNT_KDM2AB-ChIP-input_rep3


title: KDM2AB_CXXCfl_TAM_KDM2AB-ChIP-input_rep3


title: KDM2AB_CXXCfl_TAM_KDM2B_rep3

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, tamoxifen treated, KDM2B ChIP-seq


title: KDM2AB_CXXCfl_UNT_KDM2AB-ChIP-input_rep2


title: KDM2AB_CXXCfl_TAM_KDM2B_rep2


title: RR_RNA_SA_E023

description: Subcutaneous Adipocyte


title: KDM2AB_JmjCfl_UNT_H2AK119ub_rep2


title: WGBS_VA_E018

description:
E18VA
Visceral Adipocyte


title: KDM2AB_JmjCfl_UNT_H2AK119ub_rep3


title: KDM2AB_CXXCfl_TAM_KDM2B_rep1


title: KDM2AB_CXXCfl_UNT_KDM2B_rep3

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, untreated, KDM2B ChIP-seq


title: RR_RNA_SA_E013

description: Subcutaneous Adipocyte


title: RR_RNA_SA_E018

description: Subcutaneous Adipocyte


title: KDM2AB_CXXCfl_UNT_KDM2B_rep2


title: PA_RNA_VA_E023

description: Visceral Adipocyte

data_processing:
Adapters from the sequencing reads were trimmed using Trimmomatic
Reads were then mapped to the human genome (hg19) using custom bioinformatics pipeline NGSANE {Buske, 2014 #342}, which utilizes TopHat {Trapnell, 2013 #465} (TopHat v2.0.6) to perform the alignment in a strand specific manner.
Following the mapping process, htseqcount feature from the bioinformatics tool htseq {Anders, 2015 #466} was employed to count the number of uniquely mapped reads of genomic features such as genes and transcripts guided by the GENCODE GTF (v19) {Harrow, 2012 #468} annotation with the following parameter (HTSEQCOUNT_UNIQUE = 1 and GTF = “gencode.v19.annotation.gtf”)
Genome_build: hg19
Supplementary_files_format_and_content: gene_id.RPKM.csv

extract_protocol:
SA and VA samples - In brief, freshly isolated VAT and SAT samples were weighed and minced with surgical scissors. For each gram of minced tissue, 1.25 mg/mL of Collagenase I (Sigma Aldrich, CAT#C6885-1G) was added to 2 mL of HEPES buffer containing 7.9 g/L NaCl, 323 mg/L CaCl2.2H2O, 308 mg/L MgSO4.7H2O, 154.3 mg/L Na2HPO4 (anhydrous), 5 mL/L of 1M Hepes (Sigma Aldrich), 450 mg/L D-glucose, pH 7.2 – 7.4, 20 g/L BSA. The tissue was incubated for 30-45 minutes at 37ºC before being diluted 1:9 with additional HEPES buffer. The mixture was passed through a 250 micron mesh before being centrifuged at 300 rcf for 6 minutes at room temperature. The top lipid layer was removed and the adipocytes collected for snap-freezing in liquid nitrogen.; VATsamples - Snap frozen in liquid Nirtogen; PBL - 10 mL blood was collected into K2EDTA vacutainers. Following centrifugation at 1,300 rcf for 10 minutes at room temperature, the top plasma layer was removed and the buffy coat layer collected. Cells were washed three times, with vigorous resuspension, in 10 mL Tris-EDTA buffer (100 mM Tris-HCl, 0.1 mM EDTA), with pellets collected after centrifugation at 10,000 rcf for 10 minutes. The final pellet was resuspended in 500 µL of buffer and stored at -80ºC. DNA was extracted: SAVA - using proteinase K digestion and phenol chloroform extraction; VAT - DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen, Cat#69504); PBL - DNA was extracted from PBL sample types following the Gentra Puregene Blood Cell Kit (Qiagen, Cat#158445).
RNA-seq libraries were prepared using Illumina TruSeq Stranded poly-A RNA Library Prep Kit - 500 ng of RNA was used for each sample.


title: KDM2AB_CXXCfl_UNT_KDM2B_rep1


title: PA_RNA_VA_E018

description: Visceral Adipocyte


title: KDM2AB_CXXCfl_TAM_KDM2A_rep3

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, tamoxifen treated, KDM2A ChIP-seq


title: PA_RNA_SA_E023

description: Subcutaneous Adipocyte


title: KDM2AB_CXXCfl_TAM_KDM2A_rep1


title: KDM2AB_CXXCfl_TAM_KDM2A_rep2


title: KDM2AB_CXXCfl_UNT_KDM2A_rep3

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, untreated, KDM2A ChIP-seq


title: PA_RNA_SA_E013

description: Subcutaneous Adipocyte


title: RR_RNA_VA_E013

description: Visceral Adipocyte


title: RR_RNA_VA_E018

description: Visceral Adipocyte


title: KDM2AB_CXXCfl_UNT_KDM2A_rep1


title: Srp72 HET2

city: Copenhagen

first: Bo

last: Porse

address: Ole Maaløes Vej 5, 4

department: Finsen Laboratory and BRIC

country: Denmark

institute: University of Copenhagen

data_processing:
Salmon 0.12.0 was used to quantify expression of transcripts from RNA-seq experiments using mm10/GRCm38_94 Ensembl transcriptome (salmon quant -l U --seqBias --numBootstraps 30)
Genome_build: GRCm38.p6 (Ensembl release 94)
Supplementary_files_format_and_content: Tab-delimited text file including estimated number of reads mapping to each transcript and relative abundance of transcript in TPM.

source_name: LT-HSCs

extract_protocol:
sorted into RLT lysis buffer (Qiagen) and RNA was extracted using the microRNA Mini kit (Qiagen)
Ovation RNA-seq system V2 sample kit (NuGen); TruSeq V2 kit (Illumina)

treatment_protocol: FACS sorting of HSCS from Bone Marrow (Ter119-, B220-, CD3-, Mac1-, Gr1-, Sca1+, cKit+, Flt3-, CD48-, CD150+)

value: LT-HSCs

tag: mouse strain


title: Srp72 HET3


title: Srp72 HET1


title: H28815_iPSC

city: South San Francisco

first: Ittai

middle: Ethan

last: Eres

address: 750 Gateway Boulevard

department: CRADI

institute: Amgen

data_processing:
Base-calling done with RTA2 on a HiSeq 4000
Read alignment and filtering done with Hi-C User Pipeline (HiCUP, default settings) version 0.5.9 and bowtie2 version 2.2.9
Contact matrices created and normalized from Hi-C summary files produced by HiCUP version 0.5.9 with HOMER version 4.9.1 and res=10000
Genome_build: hg38 and panTro5
Supplementary_files_format_and_content: HOMER formatted and normalized contact matrices per chromosome

source_name: Induced pluripotent stem cells (iPSCs)

growth_protocol: Standard iPSC and precursor cell line conditions as described in text

extract_protocol:
Cells were formaldehyde-fixed, then pelleted and frash-frozen in N2.
In situ Hi-C libraries were prepared according to the original in situ protocol from Rao et al. 2014

tag: Sex

value: Male

value: iPSC episomally reprogrammed from fibroblast

tag: passage


title: H28126_iPSC

tag: cell type


title: H28834_iPSC


title: Srp72 WT4


title: Srp72 WT2


title: PA_RNA_VA_E013

description: Visceral Adipocyte


title: Srp72 WT3


title: TRMT2A_HEK293_iCLIP_rep3

city: Bath

first: Shobbir

last: Hussain

address: Claverton Down

department: Department of Biology and Biochemistry

institute: University of Bath

data_processing:
Raw reads where demultiplexed based on the 5' barcodes. Oligonucleotides used for reverse transcription for all libraries were synthesized to contain Unique Molecular Identifiers (UMI), thus enabling removal of PCR duplicates and the mapping of reads at the cDNA level during CLIP data processing.
CLIP data was processed using the iCount web server (http://icount.biolab.si), based on iCount Python module and associated command-line interface described on HitHub (https://github.com/tomazc/iCount).
The processed bedgraph files contains the start position of unique genome mapping reads. Each position represent accumulation of TRMT2A binding sites.
Genome_build: hg19

source_name: iCLIP HEK293 rep3

growth_protocol: HEK293 cells (ATCC) were cultured in Dulbecco’s Modified Eagles Medium (DMEM) supplemented with 10% FBS and penicillin/streptomycin. Early passage HAP1 cells (Horizon Discovery) were grown in Iscove’s Modified Dulbecco’s Medium (IMDM) supplemented with 10% FBS and penicillin/streptomycin. All cultures were maintained at a temperature of 37oC in a humidified incubator with 5% CO2. Cells were harvested by washing in Phosphate Buffered Saline (PBS) and then incubating with Trypsin-EDTA followed by further washing of detached cells in PBS.

extract_protocol:
iCLIP was performed as previously described using a UV-crosslinking based method (Konig et al. 2012). The TRMT2A antibody used for immunoprecipitation was as for FICC-Seq. Libraries containing in-line barcodes were sequenced on the Illumina HiSeq4000 platform.
Cells were harvested by washing in Phosphate Buffered Saline (PBS) and then incubating with Trypsin-EDTA followed by further washing of detached cells in PBS.

tag: method

value: iCLIP

tag: condition

value: Untreated

tag: gene symbol

tag: cell line

tag: clip antibody

tag: 5' barcode

value: NNNCAATNN

tag: 3' adapter

value: AGATCGGAAGAGCGGTTCAG


title: Srp72 WT1

tag: genotype


title: TRMT2A_HEK293_FICC-seq_rep2

description: FICC-Seq works via cellular exposure to 5-flurouracil as opposed to use of UV-mediated crosslinking

source_name: FICC-Seq HEK293

extract_protocol:
For FICC-Seq experiments, cells were treated with 100µM 5-FU (Sigma) for 24 hours and then harvested. Cell pellets were disrupted in lysis buffer consisting of 50 mM Tris-HCL pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate and treated with Turbo DNase (AM2239).  Lysates were also treated with a low concentration of RNaseI (Thermofisher AM2295 at 1:200 dilution) in order to partially fragment RNAs. Lysates were cleared by centrifugation at 13,000 r.p.m. for 15 min at 4°C and then incubated with Protein G Dynabeads (Life Technologies) in the presence of anti-TRMT2A antibody (Origene, OTI1C3). Following stringent washing, 3′-end dephosphorylation was performed with T4 PNK (New England Biolabs) before addition of a preadenylated linker using RNA ligase (New England Biolabs). 5′-end labelling was then performed using T4 PNK and 32P-ATP before protein–RNA complexes were eluted and run on Bis-Tris polyacrylamide gels. Next, nitrocellulose transfer was performed and the radioactive signal was used to dissect nitrocellulose pieces that contained TRMT2A-RNA complexes. RNA was recovered by incubating the nitrocellulose pieces in a buffer containing Proteinase K and 3.5 M urea. Reverse transcription was performed using oligonucleotides containing randomized barcodes (UMIs) and two inversely oriented adaptor regions separated by a BamHI restriction site. cDNAs were size-purified on TBE-Urea gels before being circularized by CircLigase II (Epicentre). Circularized cDNAs were then annealed to an oligonucleotide complementary to the BamHI site and then BamHI digested. Linearized cDNAs were then PCR-amplified using primers complementary to the adaptor regions using 25 cycles of PCR. Libraries containing in-line barcodes were then sequenced on the Illumina HiSeq4000 platform.
Cells were harvested by washing in Phosphate Buffered Saline (PBS) and then incubating with Trypsin-EDTA followed by further washing of detached cells in PBS.

value: FICC-Seq

value: NNNGGCGNN


title: TRMT2A_HEK293_FICC-seq_rep3

value: NNNAATGNN


title: TRMT2A_HEK293_FICC-seq_rep1


title: PA_RNA_SA_E018

description: Subcutaneous Adipocyte


title: TRMT2A_HEK293_iCLIP_rep2

source_name: iCLIP HEK293 rep2

value: NNNTTGTNN


title: H21792_iPSC

value: Female


title: KDM2AB_CXXCfl_UNT_KDM2A_rep2

tag: tissue

value: embryonic stem cells

tag: replicate

tag: treatment

value: untreated

tag: chip antibody


title: TRMT2A_HAP1_FICC-seq_rep3

source_name: FICC-Seq HAP1 rep3


title: TRMT2A_HAP1_FICC-seq_rep1

source_name: FICC-Seq HAP1 rep1

value: NNNACCTNN


title: NS_CHIP_INPUT_SF_Gata4

data_processing:
Trimming of known adapters and low-quality regions of reads was performed using Fastq-mcf (http://code.google.com/p/ea-utils), and sequence quality control was assessed using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and RSeQC (Wang, Wang, and Li 2012).
Alignment to the mm9 reference genome was performed using Bowtie 2.2.4 (Langmead and Salzberg 2012).
Peaks were called using GEM (Langmead and Salzberg 2012; Y. Guo, Mahony, and Gifford 2012).
Raw read counts per peak were generated with featureCounts (Liao, Smyth, and Shi 2014).
Counts were normalized for differences in sequencing depth between samples using upper quartile normalization separately for the the ChIP and the input samples of each transcription factor.
The normalized counts for the three transcription factor were then combined into one matrix (processed data file: "STONE_SF.ChIP_featureCounts.txt").
Genome_build: mm9
Supplementary_files_format_and_content: Text files containing normalized counts per peak matrix for all peaks from Gata4, Mef2c, and Tbx5 ChIPs combined.

source_name: neonatal mouse heart

extract_protocol:
Cells were crosslinked in 1% formaldehyde in suspension at room temperature for 10 minutes with gentle rotation. Crosslinking was quenched by addition of glycine (final 125 mM), followed by incubation at room temperature for 5 minutes with gentle rotation. Cell pellets were incubated in cell lysis buffer (20 mM Tris-HCl, pH 8, 85 mM KCl, 0.5% NP-40, protease inhibitors) for 10 minutes on a rotator at 4°C. Nuclei were isolated by centrifugation (2,500 x g, 5 minutes, 4°C), resuspended in nuclear lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, pH 8, 1% SDS, protease inhibitors) and incubated on a rotator for 30 minutes at 4°C. Chromatin was sheared using a Covaris S2 sonicator for 15 minutes (60-second cycles, 5% duty cycle, 200 cycles/burst, intensity = 5) until DNA was in the 200–700 base-pair range. Chromatin was diluted five-fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8, 167 mM NaCl, protease inhibitors) and incubated with antibody (2 mg/million cells) at 4°C overnight under rotation. Antibodies used are Santa Cruz, Gata4, sc-1237x; Cell Signal Tech, Mef2c 5030; Santa Cruz, Tbx5 (C-20) sc-17866x. Antibody-protein complexes were immunoprecipitated using Pierce Protein A/G magnetic beads at 4°C for 2 hours under rotation. Beads were washed five times (2-minute washes under rotation) with cold RIPA buffer (50 mM HEPES- KOH, pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycholate), followed by one wash in cold final wash buffer (1xTE, 50 mM NaCl). Immunoprecipitated chromatin was eluted at 65°C with agitation for 30 minutes in elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS). High-salt buffer (250 mM Tris-HCl, pH 7.5, 32.5 mM EDTA, pH 8, 1.25 M NaCl) and Proteinase K were added and crosslinking was reversed by overnight incubation at 65°C. Samples were treated with RNase A and DNA was purified with Agencourt AMPure XP beads.
Fragmented ChIP and input (whole-cell extract) DNA was end-repaired, 5′ phosphorylated and dA-tailed with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB E7645). Samples were ligated to adaptor oligos for multiplex sequencing (NEB E7335), PCR amplified, and sequenced on an Illumina NextSeq 500.

treatment_protocol: Direct cardiac reprogramming was performed as previously described (Ieda et al. 2010; Qian et al. 2013). ChIP-seq experiments were performed using an immortalized neonatal cardiac fibroblast cell line, expressing cre-excisable T-antigen, developed by Palmer Yu in the Srivastava lab. Cells were infected with freshly prepared Gata4, Mef2c, and/or Tbx5 retroviruses, and cultured with Tgfβ and Wnt inhibitors (sb431542 and xav939, respectively; Mohamed et al. 2017) prior to harvest of unsorted cells at day 2 following reprogramming initiation. We completed at least three biological replicates per condition, using 10 million cells per replicate.

tag: strain

value: alpha-MHC-GFP

value: Immortalized neonatal cardiac fibroblast cell line expressing large T antigen

tag: exogenous retroviral expression

tag: chemical treatment

tag: reprogramming time point

value: day 2


title: TRMT2A_HAP1_FICC-seq_rep2

source_name: FICC-Seq HAP1 rep2


title: TRMT2A_HAP1_iCLIP_rep_3

source_name: iCLIP HAP1 rep3


title: TRMT2A_HAP1_iCLIP_rep_2

source_name: iCLIP HAP1 rep2


title: NS_CHIP_INPUT_rep5

value: Gata4, Mef2c, Tbx5


title: NS_CHIP_INPUT_rep6


title: NS_CHIP_INPUT_rep4


title: NS_CHIP_INPUT_rep3


title: TRMT2A_HAP1_iCLIP_rep_1

source_name: iCLIP HAP1 rep1

value: NNNGGTTNN


title: NS_CHIP_INPUT_rep1


title: TRMT2A_HEK293_miCLIP_rep_2

description: miCLIP works via expression of a point mutant enzyme as opposed to use of UV-mediated crosslinking

source_name: HEK393 miCLIP rep2

extract_protocol:
For methylation-iCLIP experiments, TRMT2A expression constructs harbouring in-frame FLAG-epitope tags were used. The substrate-trapping point mutant form of TRMT2A had a glutamate residue in the catalytic domain (see Results) substituted with an alanine residue. Exponentially growing cells were transfected with cDNA constructs using Lipofectamine2000, and cells were harvested 24 hours later. Methylation-iCLIP was subsequently performed as described previously (Hussain et al. 2013a) using an anti-FLAG antibody to immunoprecipitate ectopically-expressed TRMT2A complexes. Prepared libraries containing in-line barcodes were sequenced on the Illumina HiSeq4000 platform.
Cells were harvested by washing in Phosphate Buffered Saline (PBS) and then incubating with Trypsin-EDTA followed by further washing of detached cells in PBS.

value: miCLIP

value: FLAG


title: TRMT2A_HEK293_miCLIP_rep_1

source_name: HEK393 miCLIP rep1

value: Untreated


title: NS_CHIP_INPUT_SF_Tbx5


title: TRMT2A_HEK293_iCLIP_rep1

source_name: iCLIP HEK293 rep1

value: iCLIP

value: AGATCGGAAGAGCGGTTCAG


title: NS_CHIP_INPUT_SF_Mef2c


title: Cohort C_1(9)

city: Minneapolis

first: Sabarinathan

last: Ramachandran

state: MN

address: 515 Delaware St. SE

department: Surgery

institute: University of mionnesota

data_processing:
Raw sequence that passed CASAV 1.8P/F filter were assessed by fastqc (http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
Read mapping was performed via Hisat2 (v2.0.2) using the UCSC human genome (hg38) as reference.
Cuffdiff 2.2.1 was used to quantify the expression level of each known gene in units of FPKM (fragment mapped per kilobase of exons per million mapped reads)
Differentially expressed genes were identified using the edgeR (negative binomial) feature in CLCGWB (Qiagen, Valencia, CA) using raw read counts.
Genome_build: The expressed transcripts were annotated using the nonhuman primate or Human Genome database.

organism: Macaca mulatta

source_name: Tr1 cells_with ADL infusion

growth_protocol: Not applicable

extract_protocol:
RNA was extracted using the RNAeasy Kit from Qiagen
Dual indexed Clontech Pico Mammalian stranded RNA libraries.

treatment_protocol: Peripheral blood mononuclear cells from monkeys following islet transplantation with and without ADL treatment were collected on day of sacrifice. 365 days study end point for Experimental group (with ADL infusions) and rejection for control group(no ADL infusions) and frozen at -180. Frozen aliqouts of peripheral blood monuclear cells were thawed and rested overnight in RPMI media with 10% FBS prior to antibody staining and flow sorting the next day.

tag: animal id#

value: with ADL infusion

tag: cell type

value: Tr1 cells

tag: facs-sorted population


title: Cohort C_5(21)


title: Cohort C_3(15)


title: Cohort C_2(12)


title: Cohort C_4(18)


title: NS_CHIP_SF_Tbx5_rep3

value: TBX5 goat pAb (C-20); Santa Cruz Biotechnology; sc-17866x


title: NS_CHIP_SF_Tbx5_rep2


title: Cohort B_4(26)

source_name: Tr1 cells_without ADL infusion

value: without ADL infusion


title: Cohort B_3(23)


title: Cohort B_5(34)


title: NS_CHIP_SF_Mef2c_rep1

value: Mef2c XP Rabbit mAb (D80C1); Cell Signal Tech #5030


title: NS_CHIP_SF_Mef2c_rep2


title: NS_CHIP_SF_Mef2c_rep3


title: NS_CHIP_INPUT_rep2


title: Cohort B_1(3)


title: Cohort B_2(6)


title: NS_CHIP_SF_Gata4_rep2

value: GATA-4 goat pAb (C-20); Santa Cruz Biotechnology; sc-1237x


title: NS_CHIP_SF_Gata4_rep1


title: NS_CHIP_AF_Tbx5_rep5


title: C3651_iPSC

organism: Pan troglodytes

tag: Sex

value: Female

value: iPSC episomally reprogrammed from fibroblast

tag: passage


title: NS_CHIP_SF_Tbx5_rep1

value: alpha-MHC-GFP

tag: cell line

value: Immortalized neonatal cardiac fibroblast cell line expressing large T antigen


title: NS_CHIP_AF_Tbx5_rep3

value: Gata4, Mef2c, Tbx5

value: day 2


title: C3624_iPSC

value: Male


title: NS_CHIP_AF_Mef2c_rep5


title: NS_CHIP_AF_Tbx5_rep2


title: C40300_iPSC


title: NS_CHIP_SF_Gata4_rep4


title: C3649_iPSC


title: NS_CHIP_AF_Mef2c_rep4


title: NS_CHIP_SF_Gata4_rep3


title: NS_CHIP_AF_Mef2c_rep2


title: T3 Replicate 2

city: Nijmegen

first: Aniela

last: Mundinger

address: Heyendaalseweg 135

country: Netherlands

institute: Radboud University

description:
bioreactor culture grown on nitrite
Read_counts.csv
RPKM_DESeq_Wald_statistics.csv

data_processing:
Raw reads from IonTorrent PGM sequencing were quality filtered using the CLC genomics workbench (CLC bio, Qiagen, Hilden, Germany) based on a minimum quality score of 0.05, a maximum sequencing length of 300 bp, and allowing for two ambiguous nucleotides.
Filtered reads were mapped to the N. moscoviensis genome (accession number NZ_CP011801.1) using BBMap v35.92 (Bushnell, 2015) and counted using featureCounts (Liao et al 2014) with the parameters ‘minid-0.95’ with ‘fracOverlap-0.9’ for a minimum alignment identity of 95% over 90% of the read length and ‘ambig=random’.
Differential gene expression analysis was performed using the DESeq2 package (Love et al., 2014) in R. Differential expression over multiple timepoints was tested using the likelihood ratio test. Differential expression between timepoints was tested using the Wald test. Gene expression levels were compared by ranking the CDSs based on their reads per kilobase per million reads (RPKM) values and the log2-fold to median was calculated.
Genome_build: genome accession number NZ_CP011801.1

organism: Nitrospira moscoviensis

source_name: bacterial culture

growth_protocol: Cells were grown in NOB mineral salts medium for lithoautotrophic growth as described in Spieck and Lipski 2010 with the following modifications: CaCO3 was replaced with CaCl2·2 H2O in the same concentration and the following trace element composition was used per liter of medium, 34.4 µg of MnSO4·1 H2O, 50 µg of H3BO3, 70 µg of ZnCl2, 72.6 µg of Na2MoO4·2 H2O, 20 µg of CuCl2·2 H2O, 24 µg of NiCl2·6 H2O, 80 µg of CoCl2·6 H2O, and 2000 µg of FeSO4·7 H2O.N. moscoviensis was cultivated in a 7 L glass bioreactor inoculated with an active batch culture. The bioreactor was operated at a 5 L working volume and was equipped with pH, dissolved oxygen, temperature and a level sensors, all connected to a biocontroller (bioreactor and equipment all by Applikon Biotechnology B.V., Schiedam, the Netherlands). The controller setup automatically maintained pH at 7.7 by constant flushing with Ar/CO2 (95%/5% v/v) and buffering with a 1 M KHCO3 solution. Dissolved oxygen was kept at 30% by providing filtered air or N2 gas through an aeration tube with sparger. The temperature was upheld at 39 °C by using a loop type heat exchanger. CO2 levels were controlled manually. Ar/CO2 (95%/5% v/v) flow was increased whenever CO2 was no longer in excess and acidifying the medium as indicated by the use of 1 M KHCO3 solution. The medium was constantly stirred at 150 rpm with a marine style impeller. All medium and solutions were sterilized by autoclavation or sterile filtration prior to use and the reactor operated aseptically to retain the purity of the culture.

extract_protocol:
Transcriptome analyses were performed using three technical replicates for each of the three sampling points. Biomass of 70 ml culture volume was harvested by centrifugation at 20,000 g, 4 °C, 10-20 min. RNA was immediately extracted using TRIzol reagent (Thermo Fisher Scientific) following the manufacturer’s protocol with the following modifications: all incubation and centrifugation times were doubled, RNA was precipitation overnight at –20 °C and afterwards centrifuge at 12,000 g, 4 °C, 20-30 min to collect RNA, no heating step for RNA resuspension was performed. Residual genomic DNA was removed with a DNase I (Thermo Fisher Scientific) treatment following the manufacturer’s protocol. Purity of RNA was confirmed on an ethidium bromide stained 0.9% agarose gel. The MEGAclear kit (Thermo Fisher Scientific) was used following the manufacturer’s protocol to purify the RNA and remove small RNAs. RNA quantity was controlled using the Qubit RNA HS Assay Kit (Thermo Fisher Scientific). Next, ribosomal RNA was depleted using the MICROBExpress kit (Thermo Fisher Scientific) following the manufacturer’s protocol.
Transcriptome libraries were prepared with the Ion Total RNA-Seq Kit v2 (Ion Torrent by Thermo Fisher Scientific). RNA yield and size distribution were analyzed using a 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

tag: time point


title: T3 Replicate 3


title: T3 Replicate 1


title: NS_CHIP_AF_Gata4_rep6


title: T2 Replicate 2


title: NS_CHIP_AF_Gata4_rep5


title: NS_CHIP_AF_Gata4_rep4


title: NS_CHIP_AF_Mef2c_rep3


title: T1 Replicate 3


title: NS_CHIP_AF_Gata4_rep3


title: T1 Replicate 2


title: NS_CHIP_AF_Tbx5_rep4


title: T2 Replicate 1


title: T1 Replicate 1


title: NS_CHIP_AF_Gata4_rep1


title: NS_CHIP_AF_Gata4_rep2


title: Rat_kidney_FTO

city: Guangzhou

first: Zhang

last: Zhang

state: Guangdong

address: Xingangxi Roda 135#

country: China

institute: Sun Yat-sen University

data_processing:
Library strategy: Aim-seq
The raw reads were filtered adaptors by cutadapt with at least 15 bp remaining length of paired-end reads. The clean reads were mapped to the reference genome (hg19) using Hisat2.
Reads with ACA/TGT motif internal or at the end were used to identify transcriptome-wide candidate m6A sites.
The RNAfold program of ViennaRNA package was used to predict intramolecular secondary structure of each read which supported the candidate sites, and sites with the high pairing probability were discarded.
The FTO demethylation treatment was negative control. Only the sites with at least 10% methylation ratio decrease in FTO treatment were retained as m6A sites.
Genome_build: hg19,mm10,rn6
Supplementary_files_format_and_content: text format with the information of chromosome number, site loction, strand, #reads with internal ACA in MazF, #reads sum in MazF,#reads with internal ACA in FTO, #reads sum in FTO, in one or more replicates

organism: Rattus norvegicus

source_name: Rat kidney tissue

growth_protocol: Human HEK293T cell line was cultured with DMEM (Corning) supplemented with 10% (v/v) FBS (Gibco) at 37℃ in the presence of 5% CO2, and tested to be negative for mycoplasma contamination.

extract_protocol:
Total RNA from HEK293T cells was extracted using Trizol reagent (Thermo Fisher Scientific) and the mRNA was purified using Dynabeads mRNA Purification Kit (Thermo Fisher Scientific, Cat. No. 61006). The mRNA of human tissues (brain, liver and kidney), mouse tissues (brain, liver, heart, testis and kidney) and rat tissues (brain, liver and kidney) were purchased from Takara Bio.
mRNA samples were heated to denature and digested into fragments by MazF (Takara) at 37℃ for 30 min. The fragmented mRNA was purified and end-repaired using T4 polynucleotide kinase at 37℃ for 30 min. The sample was purified for small RNA library construction.
Parallel samples demethylated by FTO in advance were as the negative controls. The FTO treatment was in 20 ul of reaction mixture containing 100-200 ng mRNA, 2.5 ug FTO demethylase, 283 μM of (NH4)2Fe(SO4)2·6H2O, 300 μM of α-KG, 2 mM of L-ascorbic acid, 20 U RNase inhibitor and 50 mM of Tris·HCl buffer (pH 7.5). After incubating at room temperature for 3h, the reaction was stopped by adding 40 mM EDTA.
Small RNA library construction protocol

value: kidney

value: FTO-MazF


title: Rat_kidney_MazF

value: MazF


title: Rat_liver_MazF

source_name: Rat liver tissue

value: liver


title: KDM2B_CXXCfl_TAM_gDNA_rep3

description: input

data_processing:
Reads were aligned to concatenated mm10 and dm6 genomes using Bowtie 2 with the ‘-- no-mixed’ and ‘-- no-discordant’ options (Langmead and Salzberg 2012). Reads that were mapped more than once were discarded, PCR duplicates were removed using SAMTools (Li et al. 2009). Reads that mapped to a custom ‘blacklist’ of genomic regions with artificially high counts, including mitochondrial DNA sequences, were also discarded.
To internally calibrate ATAC-seq, we spiked a fixed number of Drosophila SG4 cells into each sample. For data visualisation, mm10 reads were randomly subsampled by a factor that reflects the total number of spike-in reads in the same sample, as previously described (Bonhoure et al. 2014; Orlando et al. 2014; Hu et al. 2015). To account for any variation in the exact spike-in cell: mESC mixing ratio between biological replicates, the subsampling factors were additionally corrected according to the ratio of dm6/mm10 total read counts in the matched input sample.
DESeq2 (Love et al. 2014) was used with a custom R script to identify significant changes in the accessibility of gene promoters. In order to calibrate to the spike-in genome, Drosophila reads were first pre-normalised according to the exact dm6/mm10 spike-in ratio derived from the matched input gDNA sample. Drosophila read counts were then generated for a set of unique dm6 refGene gene promoters and used to calculate DESeq2 size factors. These size factors were supplied for DESeq2 normalisation of raw mm10 read counts for a custom non-redundant mm10 gene promoter set (20,633 genes). P-adj < 0.05 and fold change > 1.4 thresholds were used to determine significant changes.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files generated using the pileup function from MACS2 (Zhang et al. 2008). These represent genome coverage for merged biological replicates which have been normalised using spike-in downsampling factors.
Supplementary_files_format_and_content: DESeq2 results tables with normalised read counts, estimated log2 fold changes and statistical parameters of differential analysis.

source_name: Mouse embryonic stem cells with Drosophila SG4 cell spike-in, tamoxifen treated, gDNA-seq

growth_protocol: Mouse embryonic stem cells were cultured on gelatine-coated dishes at 37oC and 5% CO2 in Dulbecco’s Modified Eagle Medium (Life Technologies) supplemented with 15% fetal bovine serum (Labtech), 2mM L-glutamine (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), 1x non-essential amino acids (Life Technologies), 1x penicillin-streptomycin (Life Technologies), and 10 ng/ml leukemia-inhibitory factor. Drosophila S2 (SG4) cells were grown adhesively at 25oC in Schneider’s Drosophila Medium (Life Technologies), supplemented with 1x penicillin-streptomycin (Life Technologies) and 10% heat-inactivated fetal bovine serum (Labtech).

extract_protocol:
Chromatin accessibility was assayed using an adaptation of the assay for transposase accessible-chromatin (ATAC)-seq (Buenrostro et al. 2013) as previously described (King and Klose 2017), using 5x10^5 nuclei. Genomic DNA was also purified from an aliquot of the same preparation of nuclei by phenol-chloroform extraction and tagmented with Tn5, to control for sequence bias of the Tn5 transposase and to determine the exact mouse/fly cell mixing ratio for each individual sample.
ATAC-seq and input gDNA libraries were prepared by PCR amplification using custom-made Illumina barcodes (Buenrostro et al. 2013) and the NEBNext High-Fidelity 2X PCR Master Mix (NEB). Libraries were purified with two rounds of Agencourt AMPure XP bead cleanup (Agencourt, 1.5X bead:sample ratio), and were sequenced using the Illumina NextSeq 500 platform in biological triplicate or quadruplicate using 80 bp paired-end reads.

treatment_protocol: mESC lines were treated with 800 nM 4-hydroxytamoxifen (Sigma) for 96 hours to induce KDM2 long isoform knockout (Kdm2a/b-JmjCfl/fl) or ZF-CxxC domain deletion (Kdm2a/b-CXXCfl/fl, Kdm2a-CXXCfl/fl, Kdm2b-CXXCfl/fl).

value: embryonic stem cells

tag: spike in cells

value: Drosophila SG4

value: tamoxifen


title: Rat_liver_FTO


title: KDM2B_CXXCfl_TAM_gDNA_rep1

description: input

tag: genotype


title: KDM2B_CXXCfl_TAM_gDNA_rep2

description: input

tag: tissue


title: KDM2B_CXXCfl_UNT_gDNA_rep3

description: input

source_name: Mouse embryonic stem cells with Drosophila SG4 cell spike-in, untreated, gDNA-seq

tag: replicate

value: untreated


title: Rat_brain_FTO

source_name: Rat brain tissue

value: brain

tag: treatment


title: KDM2B_CXXCfl_UNT_gDNA_rep1

description: input


title: KDM2B_CXXCfl_TAM_ATAC_rep2

source_name: Mouse embryonic stem cells with Drosophila SG4 cell spike-in, tamoxifen treated, ATAC-seq


title: KDM2B_CXXCfl_TAM_ATAC_rep3


title: KDM2B_CXXCfl_TAM_ATAC_rep1


title: KDM2B_CXXCfl_UNT_ATAC_rep3

source_name: Mouse embryonic stem cells with Drosophila SG4 cell spike-in, untreated, ATAC-seq


title: KDM2B_CXXCfl_UNT_ATAC_rep2


title: CE-M15.2-38

type: RNA

city: University Park

first: Vivek

last: Kapur

state: PA

address: 205 Wartik Lab

department: Animal Science

institute: Pennsylvania State University

description:
Raw Data Technical Replicate 1: 472
Raw Data Technical Replicate 2: 473
Raw Data Technical Replicate 3: 474

data_processing:
The Ct given in the non-normalized matrix is the average of three technical replicates for each sample.
Normalization of the data was performed by using the average of 4 housekeeping genes: ACTB, GADPH, RPL4, and HMBS
Target genes were normalized to housekeeping genes and the average of the three technical replicates was the value used for further analysis (delta Ct)
The target genes of the expermiental were compared to that of the controls (delta delta Ct)
The fold change expression between the target and the controls was determined by 2^-delta delta Ct

label: FAM-MGB

organism: Gallus gallus

source_name: Experimental

label_protocol: PCR assays were performed using the custom made OpenArray plates with the Taqman Assays available through Thermo Fisher Scientific

extract_protocol: Total RNA was extracted with RNeasy Plus Mini Kit

tag: developmental stage

value: 21 days of embryonic development

tag: breed

value: Fayoumi

tag: subline

tag: infection

value: NDV

value: lung


title: KDM2B_CXXCfl_UNT_ATAC_rep1


title: KDM2A_CXXCfl_TAM_gDNA_rep2

description: input


title: KDM2A_CXXCfl_TAM_gDNA_rep3

description: input


title: KDM2A_CXXCfl_TAM_gDNA_rep1

description: input


title: CE-M15.2-37

description:
Raw Data Technical Replicate 1: 469
Raw Data Technical Replicate 2: 470
Raw Data Technical Replicate 3: 471


title: CE-M15.2-39

description:
Raw Data Technical Replicate 1: 475
Raw Data Technical Replicate 2: 476
Raw Data Technical Replicate 3: 477


title: CE-M15.2-40

description:
Raw Data Technical Replicate 1: 478
Raw Data Technical Replicate 2: 479
Raw Data Technical Replicate 3: 480


title: CE-M15.2-35

description:
Raw Data Technical Replicate 1: 463
Raw Data Technical Replicate 2: 464
Raw Data Technical Replicate 3: 465


title: CE-M15.2-34

description:
Raw Data Technical Replicate 1: 460
Raw Data Technical Replicate 2: 461
Raw Data Technical Replicate 3: 462


title: CE-M15.2-33

description:
Raw Data Technical Replicate 1: 457
Raw Data Technical Replicate 2: 458
Raw Data Technical Replicate 3: 459


title: T2 Replicate 3


title: KDM2A_CXXCfl_UNT_gDNA_rep3

description: input


title: KDM2B_CXXCfl_UNT_gDNA_rep2

description: input


title: Rat_brain_MazF


title: KDM2A_CXXCfl_UNT_gDNA_rep1

description: input


title: KDM2A_CXXCfl_TAM_ATAC_rep3


title: CE-M15.2-31

description:
Raw Data Technical Replicate 1: 451
Raw Data Technical Replicate 2: 452
Raw Data Technical Replicate 3: 453


title: CE-M15.2-28

description:
Raw Data Technical Replicate 1: 442
Raw Data Technical Replicate 2: 443
Raw Data Technical Replicate 3: 444


title: KDM2A_CXXCfl_TAM_ATAC_rep1


title: KDM2A_CXXCfl_UNT_ATAC_rep3


title: CE-M15.2-29

description:
Raw Data Technical Replicate 1: 445
Raw Data Technical Replicate 2: 446
Raw Data Technical Replicate 3: 447


title: CE-M15.2-26

description:
Raw Data Technical Replicate 1: 436
Raw Data Technical Replicate 2: 437
Raw Data Technical Replicate 3: 438


title: CE-M15.2-32

description:
Raw Data Technical Replicate 1: 454
Raw Data Technical Replicate 2: 455
Raw Data Technical Replicate 3: 456


title: KDM2A_CXXCfl_TAM_ATAC_rep2


title: KDM2AB_CXXCfl_TAM_gDNA_rep3

description: input


title: CE-M15.2-36

description:
Raw Data Technical Replicate 1: 466
Raw Data Technical Replicate 2: 467
Raw Data Technical Replicate 3: 468


title: CE-M15.2-22

description:
Raw Data Technical Replicate 1: 424
Raw Data Technical Replicate 2: 425
Raw Data Technical Replicate 3: 426


title: KDM2A_CXXCfl_UNT_gDNA_rep2

description: input


title: CE-M15.2-21

description:
Raw Data Technical Replicate 1: 421
Raw Data Technical Replicate 2: 422
Raw Data Technical Replicate 3: 423


title: CE-M15.2-27

description:
Raw Data Technical Replicate 1: 439
Raw Data Technical Replicate 2: 440
Raw Data Technical Replicate 3: 441


title: KDM2AB_CXXCfl_TAM_gDNA_rep2

description: input


title: KDM2A_CXXCfl_UNT_ATAC_rep1


title: CE-M15.2-25

description:
Raw Data Technical Replicate 1: 433
Raw Data Technical Replicate 2: 434
Raw Data Technical Replicate 3: 435


title: KDM2AB_CXXCfl_TAM_gDNA_rep4

description: input


title: CE-M15.2-23

description:
Raw Data Technical Replicate 1: 427
Raw Data Technical Replicate 2: 428
Raw Data Technical Replicate 3: 429


title: CE-M15.2-19

description:
Raw Data Technical Replicate 1: 415
Raw Data Technical Replicate 2: 416
Raw Data Technical Replicate 3: 417


title: CE-M15.2-20

description:
Raw Data Technical Replicate 1: 418
Raw Data Technical Replicate 2: 419
Raw Data Technical Replicate 3: 420


title: KDM2AB_CXXCfl_UNT_gDNA_rep4

description: input


title: CE-M15.2-17

description:
Raw Data Technical Replicate 1: 409
Raw Data Technical Replicate 2: 410
Raw Data Technical Replicate 3: 411


title: KDM2AB_CXXCfl_UNT_gDNA_rep2

description: input


title: CE-M15.2-18

description:
Raw Data Technical Replicate 1: 412
Raw Data Technical Replicate 2: 413
Raw Data Technical Replicate 3: 414


title: CE-M15.2-30

description:
Raw Data Technical Replicate 1: 448
Raw Data Technical Replicate 2: 449
Raw Data Technical Replicate 3: 450


title: CE-M15.2-16

description:
Raw Data Technical Replicate 1: 406
Raw Data Technical Replicate 2: 407
Raw Data Technical Replicate 3: 408


title: KDM2AB_CXXCfl_TAM_gDNA_rep1

description: input


title: KDM2A_CXXCfl_UNT_ATAC_rep2


title: CE-M15.2-13

description:
Raw Data Technical Replicate 1: 397
Raw Data Technical Replicate 2: 398
Raw Data Technical Replicate 3: 399


title: CE-M15.2-24

description:
Raw Data Technical Replicate 1: 430
Raw Data Technical Replicate 2: 431
Raw Data Technical Replicate 3: 432


title: KDM2AB_CXXCfl_TAM_ATAC_rep1


title: CE-M15.2-12

description:
Raw Data Technical Replicate 1: 394
Raw Data Technical Replicate 2: 395
Raw Data Technical Replicate 3: 396


title: KDM2AB_CXXCfl_TAM_ATAC_rep4


title: KDM2AB_CXXCfl_UNT_gDNA_rep3

description: input


title: KDM2AB_CXXCfl_TAM_ATAC_rep3


title: KDM2AB_CXXCfl_UNT_ATAC_rep3


title: CE-M15.2-14

description:
Raw Data Technical Replicate 1: 400
Raw Data Technical Replicate 2: 401
Raw Data Technical Replicate 3: 402


title: CE-M15.2-15

description:
Raw Data Technical Replicate 1: 403
Raw Data Technical Replicate 2: 404
Raw Data Technical Replicate 3: 405


title: KDM2AB_CXXCfl_TAM_ATAC_rep2


title: CE-M15.2-9

description:
Raw Data Technical Replicate 1: 385
Raw Data Technical Replicate 2: 386
Raw Data Technical Replicate 3: 387

source_name: Control

value: Control


title: CE-M15.2-11

description:
Raw Data Technical Replicate 1: 391
Raw Data Technical Replicate 2: 392
Raw Data Technical Replicate 3: 393


title: KDM2AB_CXXCfl_UNT_ATAC_rep2


title: CE-M15.2-10

description:
Raw Data Technical Replicate 1: 388
Raw Data Technical Replicate 2: 389
Raw Data Technical Replicate 3: 390


title: KDM2AB_CXXCfl_UNT_gDNA_rep1

description: input


title: CE-M15.2-8

description:
Raw Data Technical Replicate 1: 382
Raw Data Technical Replicate 2: 383
Raw Data Technical Replicate 3: 384


title: KDM2AB_CXXCfl_UNT_ATAC_rep1


title: CE-M15.2-7

description:
Raw Data Technical Replicate 1: 379
Raw Data Technical Replicate 2: 380
Raw Data Technical Replicate 3: 381


title: CE-M15.2-6

description:
Raw Data Technical Replicate 1: 376
Raw Data Technical Replicate 2: 377
Raw Data Technical Replicate 3: 378


title: KDM2AB_JmjCfl_TAM_gDNA_rep3

description: input


title: CE-M15.2-5

description:
Raw Data Technical Replicate 1: 373
Raw Data Technical Replicate 2: 374
Raw Data Technical Replicate 3: 375


title: KDM2AB_JmjCfl_TAM_gDNA_rep2

description: input


title: KDM2AB_JmjCfl_TAM_gDNA_rep1

description: input


title: KDM2AB_CXXCfl_UNT_ATAC_rep4


title: KDM2AB_JmjCfl_UNT_gDNA_rep3

description: input


title: CE-M15.2-3

description:
Raw Data Technical Replicate 1: 367
Raw Data Technical Replicate 2: 368
Raw Data Technical Replicate 3: 369


title: CE-M15.2-4

description:
Raw Data Technical Replicate 1: 370
Raw Data Technical Replicate 2: 371
Raw Data Technical Replicate 3: 372


title: KDM2AB_JmjCfl_UNT_gDNA_rep2

description: input


title: CE-M15.2-2

description:
Raw Data Technical Replicate 1: 364
Raw Data Technical Replicate 2: 365
Raw Data Technical Replicate 3: 366


title: KDM2AB_JmjCfl_UNT_gDNA_rep1

description: input


title: CE-M5.1-39

description:
Raw Data Technical Replicate 1: 355
Raw Data Technical Replicate 2: 356
Raw Data Technical Replicate 3: 357


title: KDM2AB_JmjCfl_TAM_ATAC_rep3


title: KDM2AB_JmjCfl_TAM_ATAC_rep1


title: CE-M5.1-37

description:
Raw Data Technical Replicate 1: 349
Raw Data Technical Replicate 2: 350
Raw Data Technical Replicate 3: 351


title: KDM2AB_JmjCfl_UNT_ATAC_rep3


title: CE-M5.1-36

description:
Raw Data Technical Replicate 1: 346
Raw Data Technical Replicate 2: 347
Raw Data Technical Replicate 3: 348


title: CE-M15.2-1

description:
Raw Data Technical Replicate 1: 361
Raw Data Technical Replicate 2: 362
Raw Data Technical Replicate 3: 363


title: CE-M5.1-38

description:
Raw Data Technical Replicate 1: 352
Raw Data Technical Replicate 2: 353
Raw Data Technical Replicate 3: 354


title: KDM2AB_JmjCfl_UNT_ATAC_rep2


title: KDM2AB_JmjCfl_UNT_ATAC_rep1


title: CE-M5.1-34

description:
Raw Data Technical Replicate 1: 340
Raw Data Technical Replicate 2: 341
Raw Data Technical Replicate 3: 342


title: CE-M5.1-35

description:
Raw Data Technical Replicate 1: 343
Raw Data Technical Replicate 2: 344
Raw Data Technical Replicate 3: 345


title: KDM2AB_JmjCfl_TAM_input_rep1

data_processing:
For RNAPII, KDM2A and KDM2B  cChIP-seq reads were aligned to concatenated mm10 and hg19 genomes and for histone ChIP-seq reads were aligned to the mouse mm10 genome, using Bowtie 2 with the ‘-- no-mixed’ and ‘-- no-discordant’ options (Langmead and Salzberg 2012). Reads that were mapped more than once were discarded, and PCR duplicates were removed using SAMTools (Li et al. 2009).
To internally calibrate RNAPII, KDM2A and KDM2B cChIP-seq, we spiked a fixed number of HEK293T cells into each sample. For data visualisation and annotation of genomic intervals, mm10 reads were randomly subsampled by a factor that reflects the total number of spike-in reads in the same sample, as previously described (Bonhoure et al. 2014; Orlando et al. 2014; Hu et al. 2015). To account for any variation in the exact spike-in cell: mESC mixing ratio between biological replicates, the subsampling factors were additionally corrected according to the ratio of hg19/mm10 total read counts in the matched input sample.
For each condition, biological replicates were merged for downstream applications. Genome coverage tracks were generated using the pileup function from MACS2 (Zhang et al. 2008). Bigwig tracks of H3K36me2 normalised to H3 were generated from merged bigwig files using the deepTools bigwigCompare function with ‘--operation ratio’ setting (Ramírez et al. 2016).
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files representing genome coverage for merged replicates.

source_name: Mouse embryonic stem cells, tamoxifen treated, gDNA-seq

extract_protocol: For histone H3 and H3K36me2 ChIP, 1x107 mESCs were crosslinked for 10 min in 1% formaldehyde. Reactions were quenched by addition of 125 mM glycine. The released nuclei were washed twice in PBS, then resuspended in lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH 8.1 and 1xPIC) and incubated on ice for 30 min. For KDM2A/B ChIP, 5x107 mESCs were resuspended in PBS and mixed with 2x106 HEK293T cells. Cells were crosslinked in 2 mM disuccinimidyl glutarate (Thermo Scientific) for 45 min at 25°C with gentle rotation, then in 1% formaldehyde for 12.5 min (methanol-free, Life Technologies). Reactions were quenched by addition of 125 mM glycine, and crosslinked cells were resuspended in lysis buffer (50mM HEPES-KOH pH 7.9, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% TritonX-100 and 1x PIC) and rotated for 10 min at 4°C. The released nuclei were washed (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA and 1x PIC) for 5 min at 4°C, and the nuclear pellet resuspended in 1 ml sonication buffer (10mM Tris-HCl pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine and 1xPIC). For RNAPII ChIP, 5x107 mESCs were resuspended in PBS and mixed with 4x106 HEK293T cells. Cells were crosslinked for 10 min in 1% formaldehyde. Reactions were quenched by addition of 150 mM glycine, and the crosslinked cells resuspended in FA-lysis buffer for 10 min (50mM HEPES pH 7.9, 150mM NaCl, 2mM EDTA, 0.5mM EGTA, 0.5% NP40, 0.1% sodium deoxycholate, 0.1% SDS, 10mM NaF, 1mM AEBSF, 1xPIC(Roche)).  For H3, H3K36me2, KDM2A/B or RNAPII ChIP, chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode), shearing genomic DNA to approximately 0.5 kb. Sonicated chromatin was diluted 10-fold in ChIP dilution buffer (1% TritonX-100, 1mM EDTA, 20mM TrisHCl pH 8, 150mM NaCl and 1x PIC) for H3, H3K36me2 or KDM2A/B ChIP, or in FA-lysis buffer for RNAPII ChIP. Chromatin was pre-cleared for 1 hour with either protein A agarose beads (Repligen, for H3, H3K36me2 or RNAPII ChIP) or protein A magnetic Dynabeads (Invitrogen, for KDM2A/B ChIP) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA. For each ChIP reaction, 150 ?g chromatin (KDM2A/B), 300 ?g chromatin (RNAPII) or chromatin corresponding to 1x105 cells (H3 or H3K36me2 ChIP) was incubated overnight with the appropriate antibody: anti-H3 (in house, 15 ?l), anti-H3K36me2 (in house, 15 ?l), anti-KDM2A (in house, 2.4 ?l), anti-KDM2B (in house, 2 ?l), anti-Rbp1-NTD (CST D8L4Y, 15 ?l), anti-Rbp1-CTD-Ser5P (CST D9N5I, 12.5 ?l), anti-Rbp1-CTD-Ser2P (CST E1Z3G, 12.5 ?l). Antibody-bound chromatin was isolated using blocked protein A agarose (H3, H3K63me2 or RNAPII ChIP) or magnetic beads (KDM2A/B ChIP) for 2 hours at 4°C. For H3, H3K36me2 or KDM2A/B ChIP, washes were performed with low salt buffer (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris-HCl pH 8, 150mM NaCl), high salt buffer (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM TrisHCl pH 8, 500mM NaCl), LiCl buffer (250mM LiCl, 1% NP40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCl pH 8) and two washes with TE buffer (10mM Tris-HCl pH 8, 1mM EDTA). For RNAPII ChIP, washes were performed with FA-Lysis buffer, FA-Lysis buffer containing 500mM NaCl, DOC buffer (250mM LiCl, 0.5% NP40, 0.5% sodium deoxycholate, 2mM EDTA, 10mM Tris-HCl pH 8) and two washes with TE buffer. ChIP DNA was eluted in elution buffer (1% SDS, 100mM NaHCO3) and crosslinks reversed overnight at 65°C with 200mM NaCl and 2 ?l RNase A (Sigma). A matched input sample (corresponding to 10% of original ChIP reaction) was treated identically. The following day, samples were treated with 20 ?g/ml Proteinase K (Sigma) for 2 hours at 45°C and purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research). For H2AK119ub1 and H3K27me3 native ChIP-seq, 5 x 107 mouse ESCs were resuspended in 1ml RSB (10mM Tris-HCl pH 8.0, 10mM NaCl, 3mM MgCl2, 5mM N-ethylmaleimide (NEM)), then lysed by addition of 28 ml RSB containing 0.1% NP40. The released nuclei were washed in RSC buffer (RSB supplemented with 0.25M sucrose and 3mM CaCl2), then resuspended in 1 ml RSC buffer containing  10mM NEM and 1xPIC(Roche). Each sample was then incubated with 150 units of MNase (Fermentas) at 37°C for 5 min, then 4 mM EDTA was added to halt MNase digestion. Following centrifugation at 5000 rpm for 5 min at 4°C, the supernatant (S1) was retained. The pellet was resuspended in 300 µl nucleosome release buffer (10mM Tris-HCl pH 7.5, 10mM NaCl, 0.2mM EDTA, 10mM NEM, 1xPIC), incubated at 4°C for 1 h, then passed five times through a 27G needle using a 1 ml syringe. After centrifugation at 5000 rpm for 5 min at 4°C, the second supernatant (S2) was combined with S1. A small aliquot of S1/S2 DNA was purified and visualized on a 1.5% agarose gel to confirm digestion to predominantly mono-nucleosomes. S1/S2 was diluted 10-fold in native ChIP incubation buffer (70mM NaCl, 10mM Tris-HCl pH 7.5, 2mM MgCl2, 2mM EDTA, 0.1% TritonX-100, 10mM NEM, 1xPIC). For each ChIP reaction, 1 ml of diluted nucleosomes was incubated overnight at 4°C with 5 ?l of anti-H2AK119ub1 (CST D27C4) or anti-H3K27me3 (in-house). Antibody-bound nucleosomes were isolated by incubation for 1 hour at 4°C with protein A agarose beads, which were pre-blocked overnight in native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA. The beads were then washed four times with native wash buffer (20mM Tris-HCl pH 7.5, 2mM EDTA, 125mM NaCl, 0.1% TritonX-100) and once with TE buffer. ChIP DNA was eluted using 100 ul of elution buffer (1% SDS, 0.1 M NaHCO3), then purified using the ChIP DNA Clean and Concentrator Kit (Zymo Research). DNA from a matched input sample (corresponding to 10% of original ChIP reaction) was also purified.

tag: chip antibody

value: none (input)


title: CE-M5.1-32

description:
Raw Data Technical Replicate 1: 334
Raw Data Technical Replicate 2: 335
Raw Data Technical Replicate 3: 336


title: KDM2AB_JmjCfl_TAM_input_rep3


title: CE-M5.1-40

description:
Raw Data Technical Replicate 1: 358
Raw Data Technical Replicate 2: 359
Raw Data Technical Replicate 3: 360


title: KDM2AB_JmjCfl_TAM_input_rep2


title: KDM2AB_JmjCfl_TAM_ATAC_rep2


title: KDM2AB_JmjCfl_UNT_input_rep3

source_name: Mouse embryonic stem cells, untreated, gDNA-seq


title: CE-M5.1-31

description:
Raw Data Technical Replicate 1: 331
Raw Data Technical Replicate 2: 332
Raw Data Technical Replicate 3: 333


title: CE-M5.1-30

description:
Raw Data Technical Replicate 1: 328
Raw Data Technical Replicate 2: 329
Raw Data Technical Replicate 3: 330


title: KDM2AB_JmjCfl_UNT_input_rep2


title: CE-M5.1-33

description:
Raw Data Technical Replicate 1: 337
Raw Data Technical Replicate 2: 338
Raw Data Technical Replicate 3: 339


title: CE-M5.1-27

description:
Raw Data Technical Replicate 1: 319
Raw Data Technical Replicate 2: 320
Raw Data Technical Replicate 3: 321


title: CE-M5.1-29

description:
Raw Data Technical Replicate 1: 325
Raw Data Technical Replicate 2: 326
Raw Data Technical Replicate 3: 327


title: KDM2AB_JmjCfl_UNT_input_rep1


title: KDM2AB_JmjCfl_TAM_H3K36me2_rep3

source_name: Mouse embryonic stem cells, tamoxifen treated, H3K36me2 ChIP-seq


title: CE-M5.1-28

description:
Raw Data Technical Replicate 1: 322
Raw Data Technical Replicate 2: 323
Raw Data Technical Replicate 3: 324


title: CE-M5.1-23

description:
Raw Data Technical Replicate 1: 307
Raw Data Technical Replicate 2: 308
Raw Data Technical Replicate 3: 309


title: KDM2AB_JmjCfl_TAM_H3K36me2_rep2


title: CE-M5.1-26

description:
Raw Data Technical Replicate 1: 316
Raw Data Technical Replicate 2: 317
Raw Data Technical Replicate 3: 318


title: KDM2AB_JmjCfl_TAM_H3_rep3

source_name: Mouse embryonic stem cells, tamoxifen treated, H3 ChIP-seq


title: KDM2AB_JmjCfl_TAM_H3K36me2_rep1


title: CE-M5.1-25

description:
Raw Data Technical Replicate 1: 313
Raw Data Technical Replicate 2: 314
Raw Data Technical Replicate 3: 315


title: KDM2AB_JmjCfl_UNT_H3K36me2_rep3

source_name: Mouse embryonic stem cells, untreated, H3K36me2 ChIP-seq


title: KDM2AB_CXXCfl_TAM_RNAP_input_rep4

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, tamoxifen treated, gDNA-seq

value: human HEK293T cells


title: CE-M5.1-18

description:
Raw Data Technical Replicate 1: 292
Raw Data Technical Replicate 2: 293
Raw Data Technical Replicate 3: 294


title: KDM2AB_CXXCfl_TAM_RNAP_input_rep3


title: CE-M5.1-15

description:
Raw Data Technical Replicate 1: 283
Raw Data Technical Replicate 2: 284
Raw Data Technical Replicate 3: 285


title: KDM2AB_JmjCfl_UNT_H3K36me2_rep2


title: CE-M5.1-24

description:
Raw Data Technical Replicate 1: 310
Raw Data Technical Replicate 2: 311
Raw Data Technical Replicate 3: 312


title: KDM2AB_CXXCfl_UNT_RNAP_input_rep4

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, untreated, gDNA-seq


title: KDM2AB_CXXCfl_TAM_RNAP_S5P_rep4

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, tamoxifen treated, S5P-RNAPII ChIP-seq


title: CE-M5.1-9

description:
Raw Data Technical Replicate 1: 265
Raw Data Technical Replicate 2: 266
Raw Data Technical Replicate 3: 267


title: KDM2AB_JmjCfl_UNT_H3K36me2_rep1


title: CE-M5.1-22

description:
Raw Data Technical Replicate 1: 304
Raw Data Technical Replicate 2: 305
Raw Data Technical Replicate 3: 306


title: KDM2AB_CXXCfl_TAM_RNAP_S5P_rep1


title: CE-M5.1-16

description:
Raw Data Technical Replicate 1: 286
Raw Data Technical Replicate 2: 287
Raw Data Technical Replicate 3: 288


title: KDM2AB_CXXCfl_UNT_RNAP_S5P_rep2

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, untreated, S5P-RNAPII ChIP-seq


title: CE-M5.1-3

description:
Raw Data Technical Replicate 1: 247
Raw Data Technical Replicate 2: 248
Raw Data Technical Replicate 3: 249


title: CE-M5.1-4

description:
Raw Data Technical Replicate 1: 250
Raw Data Technical Replicate 2: 251
Raw Data Technical Replicate 3: 252


title: CE-M5.1-21

description:
Raw Data Technical Replicate 1: 301
Raw Data Technical Replicate 2: 302
Raw Data Technical Replicate 3: 303


title: KDM2AB_JmjCfl_TAM_H3_rep2


title: KDM2AB_CXXCfl_TAM_RNAP_S2P_rep2

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, tamoxifen treated, S2P-RNAPII ChIP-seq


title: KDM2AB_JmjCfl_TAM_H3_rep1


title: CE-M5.1-20

description:
Raw Data Technical Replicate 1: 298
Raw Data Technical Replicate 2: 299
Raw Data Technical Replicate 3: 300


title: KDM2AB_JmjCfl_UNT_H3_rep3

source_name: Mouse embryonic stem cells, untreated, H3 ChIP-seq


title: KDM2AB_JmjCfl_UNT_H3_rep2


title: KDM2AB_CXXCfl_TAM_RNAP_input_rep2


title: CE-M5.1-17

description:
Raw Data Technical Replicate 1: 289
Raw Data Technical Replicate 2: 290
Raw Data Technical Replicate 3: 291


title: CE-M5.1-19

description:
Raw Data Technical Replicate 1: 295
Raw Data Technical Replicate 2: 296
Raw Data Technical Replicate 3: 297


title: CE-M5.1-14

description:
Raw Data Technical Replicate 1: 280
Raw Data Technical Replicate 2: 281
Raw Data Technical Replicate 3: 282


title: KDM2AB_JmjCfl_UNT_H3_rep1


title: KDM2AB_CXXCfl_TAM_RNAP_input_rep1


title: CE-M5.1-13

description:
Raw Data Technical Replicate 1: 277
Raw Data Technical Replicate 2: 278
Raw Data Technical Replicate 3: 279


title: CE-M5.1-12

description:
Raw Data Technical Replicate 1: 274
Raw Data Technical Replicate 2: 275
Raw Data Technical Replicate 3: 276


title: KDM2AB_CXXCfl_UNT_RNAP_input_rep3


title: KDM2AB_CXXCfl_UNT_RNAP_S2P_rep3

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, untreated, S2P-RNAPII ChIP-seq


title: CE-Ghs13-39

description:
Raw Data Technical Replicate 1: 235
Raw Data Technical Replicate 2: 236
Raw Data Technical Replicate 3: 237

value: Leghorn


title: KDM2AB_CXXCfl_UNT_RNAP_S2P_rep2


title: CE-M5.1-11

description:
Raw Data Technical Replicate 1: 271
Raw Data Technical Replicate 2: 272
Raw Data Technical Replicate 3: 273


title: KDM2AB_CXXCfl_UNT_RNAP_input_rep2


title: KDM2AB_CXXCfl_TAM_RNAP_S5P_rep2


title: CE-M5.1-10

description:
Raw Data Technical Replicate 1: 268
Raw Data Technical Replicate 2: 269
Raw Data Technical Replicate 3: 270


title: KDM2AB_CXXCfl_UNT_RNAP_S5P_rep4


title: KDM2AB_CXXCfl_UNT_RNAP_input_rep1


title: CE-M5.1-7

description:
Raw Data Technical Replicate 1: 259
Raw Data Technical Replicate 2: 260
Raw Data Technical Replicate 3: 261


title: CE-M5.1-6

description:
Raw Data Technical Replicate 1: 256
Raw Data Technical Replicate 2: 257
Raw Data Technical Replicate 3: 258


title: CE-M5.1-8

description:
Raw Data Technical Replicate 1: 262
Raw Data Technical Replicate 2: 263
Raw Data Technical Replicate 3: 264


title: KDM2AB_CXXCfl_UNT_RNAP_S5P_rep1


title: CE-Ghs13-35

description:
Raw Data Technical Replicate 1: 223
Raw Data Technical Replicate 2: 224
Raw Data Technical Replicate 3: 225


title: KDM2AB_CXXCfl_TAM_RNAP_S2P_rep3


title: KDM2AB_CXXCfl_TAM_RNAP_S2P_rep1


title: KDM2AB_CXXCfl_UNT_RNAP_NTD_rep2

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, untreated, RNAPII-NTD ChIP-seq


title: CE-Ghs13-33

description:
Raw Data Technical Replicate 1: 217
Raw Data Technical Replicate 2: 218
Raw Data Technical Replicate 3: 219


title: CE-Ghs13-40

description:
Raw Data Technical Replicate 1: 238
Raw Data Technical Replicate 2: 239
Raw Data Technical Replicate 3: 240


title: KDM2AB_CXXCfl_UNT_RNAP_S2P_rep1


title: KDM2AB_CXXCfl_UNT_RNAP_NTD_rep4


title: CE-M5.1-2

description:
Raw Data Technical Replicate 1: 244
Raw Data Technical Replicate 2: 245
Raw Data Technical Replicate 3: 246


title: CE-Ghs13-38

description:
Raw Data Technical Replicate 1: 232
Raw Data Technical Replicate 2: 233
Raw Data Technical Replicate 3: 234


title: CE-M5.1-1

description:
Raw Data Technical Replicate 1: 241
Raw Data Technical Replicate 2: 242
Raw Data Technical Replicate 3: 243


title: KDM2AB_CXXCfl_TAM_RNAP_NTD_rep2

source_name: Mouse embryonic stem cells with human HEK293T cell spike-in, tamoxifen treated, RNAPII-NTD ChIP-seq


title: KDM2AB_CXXCfl_TAM_RNAP_NTD_rep3


title: CE-M5.1-5

description:
Raw Data Technical Replicate 1: 253
Raw Data Technical Replicate 2: 254
Raw Data Technical Replicate 3: 255


title: CE-Ghs13-36

description:
Raw Data Technical Replicate 1: 226
Raw Data Technical Replicate 2: 227
Raw Data Technical Replicate 3: 228


title: KDM2AB_CXXCfl_TAM_RNAP_NTD_rep1


title: CE-Ghs13-34

description:
Raw Data Technical Replicate 1: 220
Raw Data Technical Replicate 2: 221
Raw Data Technical Replicate 3: 222