title: S2_UTD_deep_Ribo-seq

city: Leeds

first: Julie

last: Aspden

address: Woodhouse

department: FBS

institute: University of Leeds

description: ribosome footprinted RNA

data_processing:
Library strategy: Ribo-seq
Adaptor sequences were trimmed using Cutadapt (v.2.10) with minimum read length of 25bp, and untrimmed outputs retained for RNA-seq.
Low-quality reads (score <20 for 10% or more of read) were discarded using FASTQ Quality Filter, FASTX-Toolkit (v.0.0.14).
Reads aligning to rRNA and tRNA sequences were discarded using Bowtie 2 (v2.4.1) and one base was removed from the 3' end of reads.
Unaligned Ribo-seq reads and RNA-seq reads were aligned to the Drosophila melangoster reference genome (Release 6.35) using the split-aware aligner STAR (v2.7.5c). The STAR genome index was built with an sjdbOverhang of 73.
Alignments flagged as secondary alignment were filtered out with samtools (v1.10), ensuring 1 genomic position per aligned read. "samtools view -b -F 0X100"
RiboTaper was applied to these data and RNA-seq from GEO sample GSM1477474 (SRA run SRR1548660) to find ORFs and quantify the p-site positions per ORF (reported in score column of processed data bed file).
Genome_build: dmel_r6.35
Supplementary_files_format_and_content: bed file of open reading frames determined by RiboTaper pipeline.

organism: Drosophila melanogaster

source_name: S2 cells

growth_protocol: S2 cells were grown under standard conditions in Schneiders medium with 10% FBS

extract_protocol:
Footprinted lysates were loaded onto sucrose gradients and subjected to ultracentrifugation. We purified 80S ribosomes away from ribosomal subunits and polysomes. RNA was isopropanol precipitated, TURBO DNase treated, phenol:chloroform extracted and ethanol precipitated. 28-34 nt ribosome footprints were gel purified, T4 PNK treated, isopropanol precipitated.
rRNA depletion carried out with custom made beads. rRNA depleted footprints were ethanol precipitated again. Library preparation was completed with the NEBNext Multiplex Small RNA Library Prep Set for Illumina E7580S. Libraries were sequenced on NextSeq 500 Illumina sequencer with 75 bp single end protocol.

treatment_protocol: Cycloheximide treated cells were treated for 3 minutes with 100 mg/mL cycloheximide before being pelleted. All cells were pelleted, washed (1 X PBS, 100 mg/ml cycloheximide) and resuspended in high ionic lysis buffer; 50 mM Tris-HCl pH8, 150 mM NaCl, 10 mM MgCl2, 1 mM DTT, 1% NP40, 100 µg/ml cycloheximide, Turbo DNase (Invitrogen), RNasin® Plus RNase Inhibitor (Promega), cOmplete protease inhibitor (Roche). Nuclei were removed and cytoplasmic lysates were footprinted overnight at 4°C. Two different footprinting conditions: 1) Ambion RNAse I (AM2295) in high ionic lysis buffer, 2) Thermo Fisher RNAse I (EN0601) in low ionic lysis buffer (100 mM Tris-HCl pH8, 50 mM NaCl, 10 mM MgCl2).

value: S2 cells

value: untreated

value: Thermo Fisher RNAse I (EN0601)

value: low ionic lysis buffer


title: C6

status: Public on Jan 25 2022

type: SRA

city: Nijmegen

first: Jodie

middle: Anne

last: Schildkraut

address: Geert Grooteplein Zuid 10

country: Netherlands

institute: Radboudumc

library_source: transcriptomic

data_processing:
Illumina demultiplexing bcl2fastq
Mapping using STAR (v2.7.0)
Create count table with GenomicAlignments
Genome_build: NC_010397.1
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample

molecule: total RNA

organism: Mycobacteroides abscessus

source_name: Frozen reference strain

growth_protocol: Bacteria were grown to early log-phase prior to start of experiment

extract_protocol:
RNA isolation was performed using the Nucleospin RNA kit (Machery Nagel, Düren, Germany), with slight modifications. Following isolation, RNA integrity was determined, ribosomal RNA was depleted and the mRNA library was constructed and sequenced on a NextSeq 500 (Illumina, San Diego, CA).
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Bacteria were treated with antibiotica for 4 or 24 hours or left untreated

tag: treatment

value: Amikacin

tag: time

value: 24 hours


title: iDART_SMART-seq APOBEC1-YTH Cell 4

city: Durham

first: Matthew

last: Tegowski

state: NC

address: 153 Research Dr.

department: Biochemistry

country: USA

institute: Duke University

data_processing:
Alignment to hg38 and UMI deduplication using Cell Ranger 3.1.0 (10x Genomics scRNA-seq samples only)
SMART-seq2 and bulk RNAseq samples: Trimming of adaptor sequences (Flexbar 3.0.3)
SMART-seq2 and bulk RNAseq samples:  Alignment using STAR 2.7.5c, and deduplication using SAMtools (1.11) fixmate and markdup
All samples: Identification of m6A sites using Bullseye
All samples: Expression normalization and integrated clustering of cells using Seurat (3.2.1)
Genome_build: hg38
Supplementary_files_format_and_content: Bed files for bulk RNAare a list of m6A sites identified within each replicate using Bullseye. High confidence m6A sites in paper were a result of keeping only these sites that occur in at least 2 of 3 APOBEC1-YTH replicates
Supplementary_files_format_and_content: a bed file consisting of APOBEC1-mediated C-to-U editing sites identified by downstream analysis

molecule: polyA RNA

organism: Homo sapiens

source_name: Kidney

growth_protocol: HEK293T cells expressing APOBEC1-YTH or APOBEC1-YTHmut (iDART cells) were cultured in DMEM +10% FBS with penicillin/streptomycin.

extract_protocol:
SMART-seq2 cells: Single cells were sorted using a FACS Aria II (BD) directly into lysis buffer provided with the SMART-seq HT Kit (Takara, Cat. 634438). The SMART-seq HT Kit was then used for reverse transcription and cDNA amplification. Sequencing libraries were constructed from cDNA using the Nextera XT kit (Illumina, FC-131-1096) and dual Nextera-compatible CDI indexes from IDT. Samples were pooled and sequenced targeting 5,000,000 reads/library (150bp paried-end) on a Novaseq6000 using the S4 flowcell.   10X Genomics cells: A single cell suspension was prepared according to manufacturer's instruction for Chromium v3 3' end sequencing (10x Genomics, Manual Part #CG00053). Samples were sequenced targeting 80,000 reads per cell on a Novaseq6000 using the S1 flowcell.  Bulk RNA-seq: Trizol was used to extract total RNA from iDART cells. The UltraII Directional RNA Library Prep Kit (NEB, E7760) with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, 7490) was used to generate sequencing libraries using 900ng of input RNA. The samples were sequenced targeting ~30 million reads (50bp paired-end sequencing) per sample on a Novaseq6000 using the S1 flowcell.
SMART-seq2 scRNA-seq: cDNA libraries were generated using the SMART-seq HT Kit (Takara, Cat. 634438). Final libraries were prepared using Nextera XT DNA Library Prep Kit (Illumina, FC-131-1096).   10x scRNA-seq: Libraries were generated from a single cell suspension using the 10x Genomics NextGEM Single Cell 3' Kit, v3.1 chemistry (10x Genomics)  Bulk-RNA seq: The UltraII Directional RNA Library Prep Kit (NEB, E7760) with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, 7490) was used to generate sequencing libraries using 900ng of input RNA.

treatment_protocol: When cells were 60-70% confluent, iDART cells were treated with 1µg/µL doxycycline for 24 hours for bulk RNA-seq, 10x Genomics scRNA-seq, and SMART-seq2 scRNA-seq

tag: cell line

value: HEK293T Cells

tag: transgene

value: 1microg/mL doxycycline; 24 hours


title: S2_UTD_Ribo-seq

country: United Kingdom

description: ribosome footprinted RNA

tag: cell type


title: A6


title: ChIP_H3K36me3_Setdb1KO_rep1

city: Montpellier

first: Amandine

last: BARRAL

state: Hérault

address: 141 rue de la cardonille

country: France

institute: IGH - CNRS

library_source: genomic

data_processing:
Quality controls of ChIP-seq Fastq files were performed by FastQC. ChIP-seq Fastq read files are mapped to the mm10 reference genome using Bowtie2 with default parameters. To determine for ChIP-seq peaks with a broad profile, datasets were processed using MACS2 with --broad and --g mm options. H3K36me3, H3K36me2 and H3K36me1 bigWig files were normalized using drosophila spike-ins, which were mapped to dm6 reference genome using Bowtie2 with default parameters. The normalization factor was determined as described in manufactured protocol (Spike-in Normalization from Active Motif). For each sample, normalized BigWig files were generated using Deeptools bamCoverage with --bs 50, --normalizeUsing RPKM and --scaleFactor options, where scaleFactor is the normalization factor specific to each sample.
Genome_build: mm10
Supplementary_files_format_and_content: bw files correpond RPKM-normalized bigwig binary files (50bp bins), which can be visualized using the Integrative Genomics Viewer (IGV) software.

molecule: genomic DNA

organism: Mus musculus

source_name: Mouse embryonic stem cells

growth_protocol: Mouse embryonic stem cells and derivates were grown on gelatin coated dishes in DMEM containing 15% (v/v) fetal bovine serum, Penicillin streptomycin (10 000U/mL), 2mM L-Glutamine, 1X MEM Non-Essential Amino Acids, 10mM 2-Mercaptoethanol and 2mL of homemade Leukemia Inhibitory Factor (LIF) at 37°C with 5% C02. Setdb1 was knocked down by treating cells for 5 days with 800 nM 4-hydroxytamoxifen (4-OHT) in culture medium.

extract_protocol:
Two 15cm-dishes with 2.10^7 cells in 20mL of culture media were used for each ChIP-seq experiment.  ChIP-seq was performed as described in ChIP-IT High sensitivity Kit from Active Motif with a few adaptations. Chromatin is sonicated by Bioruptor: 30sec ON – 30sec OFF, 10 cycles, high power. Sonicate for a total of 40 cycles for histones and of 30 cycles for TFs with 10min break on ice every 10 cycles. For quantitative ChIP reaction drosophila spike-in from Active Motif was used. For histone methylation of lysine 36, 25µg chromatin was used with 50ng Spike-in Chromatin + 4µg antibody + 2ug Spike-in Antibody. Spike-in antibody also required a blocking step. ChIP-DNA was eluted in 36µL with pre-warmed Elution Buffer then ChIP-DNA concentration was quantified with the Qubit dsDNA HS Assay Kit. 2,5ng to 10ng of ChIP-DNA were used for library preparation.
2,5ng to 10ng ChIP-DNA were used for library preparation. ChIP-seq library preparation was performed as described in TruSeq ChIP Sample Preparation kit from Illumina with few adaptations. The Enrich DNA Fragment step is proceeded before Purify Ligation Products step. AMPure XP beads were air-dry 7min. 1µL library was diluted 1:10 in H20 to quantified library concentration by Qubit DNA HS assay quantification. Agilent High Sensitivity DNA Electrophoresis was run to check DNA fragment size. Before sequencing, ChIP-seq libraries were quantified by qPCR. Libraries were sequenced 50bp or 100bp single-reads with 50 million reads of depth sequencing on HiSeq 2000 Illumina or NovaSeq 6000 Illumina machine at the MGX platforms.

tag: strain

value: Mouse embryonic stem cells

tag: chip antibody

value: H3K36me3, Abcam ab9050

tag: spike-in

value: Drosophila spike-in chromatin


title: iDART_10xGenomics_scDART-seq_APOBEC1-YTHmut Replicate 1

description:
10x files consist of 3 fastqs (R1,R2,I1),
10xGenomics_m6Asites.bed


title: B6


title: ChIP_H3K36me3_WT_rep2

description: processed data file: Dualdomains-peak.bed


title: Bb1

value: Clarithromycin

value: 4 hours


title: S2_UTA_Ribo-seq

description: ribosome footprinted RNA

value: Ambion RNAse I (AM2295)

value: high ionic lysis buffer


title: S2_TD_Ribo-seq

description: ribosome footprinted RNA

value: treated with cycloheximide


title: ATACseq_Setdb1KO_rep1

data_processing:
Quality controls of ATAC-seq Fastq files were performed by FastQC. ATAC-seq adaptors and ATAC-seq reads were removed or trimmed using Cutadapt. Paire-end reads of ATAC-seq Fastq files were mapped to the mm10 reference genome using Bowtie2 with default parameters. Quality controls of ATAC-seq alignments were performed with ATACseqQC. To determine peaks for ATAC-seq, datasets were processed using MACS2 with default parameters and --g mm and –B options. BigWig files were generated using Deeptools bamCoverage with --bs 50 and --normalizeUsing RPKM options.
Genome_build: mm10
Supplementary_files_format_and_content: bw files correpond RPKM-normalized bigwig binary files (50bp bins), which can be visualized using the Integrative Genomics Viewer (IGV) software.

extract_protocol:
1.10^6 cells were used for each Omni-ATAC-seq experiment.  If there is 5 to 15% of death cells, cells are incubated with DNAse I at 37°C for 30min then cells are collected at 1200g for 3min at 4°C and washed 3 times with 1mL ice-cold PBS. Cells were resuspended into 1mL ice-cold PBS, 50,000 cells (50µL) were transfered into fresh 1,5mL Eppendorf tube. Omni-ATAC-seq was performed as described in Corces et al 2017, Nature Methods. Transposase enzyme was from Netera XT DNA library Preparation Kit.
Omni-ATAC-seq library preparation was performed as described in Buesnrostro et al 2013 Nature Methods with few adaptations. Illumina sequencing primers was from Buenrostro et al 2013 Nature Methods. To determine the number of additional cycles required, Ct corresponding to plateau is identified and ¼ of Ct + 1 defined the number of additional cycles. Then Omni-ATAC-seq libraries were cleaned up with AMPure XP beads to remove DNA fragment smaller than 100bp and longer than 1,5Kb. 30µL H2O was added to libraries obtain a final volume of 50µL then 25µL AMPure XP beads were added and incubate at RT for 15min. AMPure XP beads are placed on magnetic stand for 5min at RT. Supernatants were transfered into fresh tube and 65µL AMPure XP beads were added to samples then incubate at RT for 15min and place on magnetic stand for 5min at RT. Supernatant was removed. AMPure XP beads were whased with 200µL fresh 80%-EtOH twice. AMPure XP beads were air-dry for 7min at RT. 22µL Buffer EB was added to resuspend AMPure XP beads then they are incubated at RT for 2min and placed on magnetic stand for 5min at RT. 20µL ATAC-library samples were transferred into fresh 1,5mL Eppendorf tube. 1µL of library was diluted 1:100 to do qPCR quality control and to run Agilent High Sensitivity DNA Electrophoresis to check DNA fragment size. Before sequencing, ATAC-seq libraries are quantified by qPCR. Libraries were sequenced 75bp paired-end with 50 million reads of depth sequencing on HiSeq 2000 Illumina machine at the MGX platforms.

value: 5 days with 800 nM 4-hydroxytamoxifen (4-OHT)


title: iDART_10xGenomics_scDART-seq_APOBEC1-YTH Replicate 3


title: ChIP_H3K36me3_WT_rep1


title: EC_BW25113_CoIP_SpyCas9-3xF_mutant_leader_array_R2

city: Würzburg

first: Tom

last: Gräfenhan

address: Josef-Schneider-Str. 2, Bau D15

department: Core Unit SysMed

country: Germany

institute: University of Wuerzburg

description:
RNA bound to SpyCas9-3xFLAG
eluted after co-immunoprecipitation with anti-FLAG antibody

data_processing:
Base calling with Illumina NextSeq RTA v2.11.3
FASTQ conversion with bcl2fastq v2.20.0.422
FASTQ quality and adapter trimming using Cutadapt (Martin, 2011) version 2.5 (cutadapt parameters: --nextseq-trim=20 -m 1 -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC)
Alignment of reads longer than 11 nt to reference genome using segemehl version 0.2.0 with minimum accuracy 95% (parameters for reademption align: -l 12 -a 95 ) (READemption 0.4.5, Förstner et al., 2014)
Coverage calculation via reademption coverage based on all aligned reads (full-length); Normalization: coverage-tnoar_mil_normalized (counts divided by total number of aligned reads and multiplied by 1,000,000) (READemption 0.4.5, Förstner et al., 2014)
Genome_build: E. coli K-12 BW25113: RefSeq assembly GCF_000750555.1 together with NL-Tagged-SpCas9-Plasmid (native leader, https://benchling.com/s/seq-JlrK1TcQnY4F3exlry0o) or ML-Tagged-SpCas9-Plasmid (mutated leader, https://benchling.com/s/seq-YJpL5iHCvtffFQaxEwFE); L. rhamnosus GG: RefSeq assembly GCF_000026505.1 and Tagged-LrCas9-Plasmid (https://benchling.com/s/seq-n65S4AdjwUw640uzpcNU)
Supplementary_files_format_and_content: wiggle files containing positional read coverage values

organism: Escherichia coli

source_name: E. coli BW25113 cells

growth_protocol: E. coli cells were grown at 37°C in Luria Bertani (LB) broth (5  g/L NaCl, 5 g/L yeast extract, 10 g/L tryptone) with shaking at 250 rpm. The antibiotic kanamycin was added at 50 µg/mL to maintain plasmids. L. rhamnosus cells were grown at 37°C in De Man, Rogosa and Sharpe (MRS) broth (Becton Dickinson) without agitation. The antibiotic chloramphenicol was added at 10 µg/mL to maintain plasmids.

extract_protocol:
Briefly, the cells harboring corresponding plasmids were lysed, tagged proteins were enriched through immunoprecipitation, and RNA bound to the tagged proteins were extracted using phenol-chloroform-isoamyl alcohol method for RIP-seq. The cells harboring the plasmids encoding untagged proteins were subjected to the same extraction procedures to use as controls.
The extracted RNA was treated with DNase I (Thermo Scientific, EN0525) following the manufacturer’s instructions. cDNA libraries for Illumina sequencing were constructed by Vertis Biotechnologie AG, Germany (http://www.vertis-biotech.com). Briefly, the resulting RNA was subjected to oligonucleotide adapter ligation on the 3′ end, first-strand cDNA synthesis using M-MLV reverse transcriptase (Agilent), and Illumina TruSeq sequencing adapter ligation on the 3′ end. The resulting cDNA was PCR-amplified using Herculase II Fusion DNA Polymerase (Agilent) with 13 amplification cycles following the manufacturer’s instructions, purified using Agencourt AMPure XP kit (Beckman Coulter Genomics) following the manufacturer’s instructions, and analyzed by capillary Electrophoresis.

treatment_protocol: No treatment applied during growth. Bacterial lysates from biological replicates were used for coIP.

tag: crispr array leader

value: mutant leader

tag: antibody for coip

value: ANTI-FLAG M2 (Sigma, cat. #F 1804)

tag: replicate


title: ChIP_RNApolII_Setdb1KO_rep2

data_processing:
Quality controls of ChIP-seq Fastq files were performed by FastQC. ChIP-seq Fastq read files are mapped to the mm10 reference genome using Bowtie2 with default parameters. To determine for ChIP-seq peaks with a narrow profile, datasets were processed using MACS2 with default parameters and --g mm option. For profiles characterized by enrichments in broad regions, datasets were processed using MACS2 with --broad and --g mm options. BigWig files were generated using Deeptools bamCoverage with --bs 50 and --normalizeUsing RPKM options.
Genome_build: mm10
Supplementary_files_format_and_content: bw files correpond RPKM-normalized bigwig binary files (50bp bins), which can be visualized using the Integrative Genomics Viewer (IGV) software.

extract_protocol:
Two 15cm-dishes with 2.10^7 cells in 20mL of culture media were used for each ChIP-seq experiment.  ChIP-seq was performed as described in ChIP-IT High sensitivity Kit from Active Motif with a few adaptations. Chromatin is sonicated by Bioruptor: 30sec ON – 30sec OFF, 10 cycles, high power. Sonicate for a total of 40 cycles for histones and of 30 cycles for TFs with 10min break on ice every 10 cycles. For ChIP reaction, 20µg chromatin was used with 4µg antibody for histone and 30µg chromatin + 4µg antibody for none-histone proteins. ChIP-DNA was eluted in 36µL with pre-warmed Elution Buffer then ChIP-DNA concentration was quantified with the Qubit dsDNA HS Assay Kit. 2,5ng to 10ng of ChIP-DNA were used for library preparation.
2,5ng to 10ng ChIP-DNA were used for library preparation. ChIP-seq library preparation was performed as described in TruSeq ChIP Sample Preparation kit from Illumina with few adaptations. The Enrich DNA Fragment step is proceeded before Purify Ligation Products step. AMPure XP beads were air-dry 7min. 1µL library was diluted 1:10 in H20 to quantified library concentration by Qubit DNA HS assay quantification. Agilent High Sensitivity DNA Electrophoresis was run to check DNA fragment size. Before sequencing, ChIP-seq libraries were quantified by qPCR. Libraries were sequenced 50bp or 100bp single-reads with 50 million reads of depth sequencing on HiSeq 2000 Illumina or NovaSeq 6000 Illumina machine at the MGX platforms.

value: Pol II (F-12), Santa Cruz sc-55492


title: iDART_10xGenomics_scDART-seq_APOBEC1-YTH Replicate 2


title: S2_TA_Ribo-seq

description: ribosome footprinted RNA

value: S2 cells


title: Ab1


title: C2


title: iDART_10xGenomics_scDART-seq_APOBEC1-YTH Replicate 1


title: ChIP_RNApolII_Setdb1KO_rep1


title: A2


title: B2


title: EC_BW25113_CoIP_SpyCas9_mutant_leader_array_R2

description:
RNA bound non-specifically
eluted after co-immunoprecipitation with anti-FLAG antibody


title: Bulk_iDART_APOBEC1-YTHmut Replicate 3

description:
Bed file for bulk YTHmut DART-seq samples consists of all RAC sites 10%-100% (all three YTHmut replicates averaged)
Bulk_YTHmut_RACsites.bed


title: ChIP_RNApolII_WT_rep2


title: Bulk_iDART_APOBEC1-YTHmut Replicate 2


title: ChIP_RNApolII_WT_rep1


title: Cb4

value: Growth Control


title: Cb1


title: Bulk_iDART_APOBEC1-YTH Replicate 3

description:
Bed files for bulk DART-seq are sites found in each replicate. High confidence sites were found by further filtering for sites found in at least 2 of 3 replicates. Supplied BAM files have PCR duplicates removed using SAMtools markdup/fixmate
WTC12_C_m6Asites.bed


title: Bulk_iDART_APOBEC1-YTHmut Replicate 1


title: EC_BW25113_CoIP_SpyCas9_native_leader_array_R2

value: native leader


title: EC_BW25113_CoIP_SpyCas9-3xF_mutant_leader_array_R1


title: ChIP_H3K4me3_Setdb1KO_rep1

value: H3K4me3, Abcam ab8580


title: ChIP_H3K4me3_WT_rep1


title: Ab4


title: EC_BW25113_CoIP_SpyCas9_mutant_leader_array_R1


title: Bb4


title: ChIP_H3K9me3_Setdb1KO_rep2

value: H3K9me3, Abcam ab8898


title: Bulk_iDART_APOBEC1-YTH Replicate 2

description:
Bed files for bulk DART-seq are sites found in each replicate. High confidence sites were found by further filtering for sites found in at least 2 of 3 replicates. Supplied BAM files have PCR duplicates removed using SAMtools markdup/fixmate
WTC12_B_m6Asites.bed

value: HEK293T Cells

value: 1microg/mL doxycycline; 24 hours


title: C7

value: 24 hours


title: Bulk_iDART_APOBEC1-YTH Replicate 1

description:
Bed files for bulk DART-seq are sites found in each replicate. High confidence sites were found by further filtering for sites found in at least 2 of 3 replicates. Supplied BAM files have PCR duplicates removed using SAMtools markdup/fixmate
WTC12_A_m6Asites.bed


title: ChIP_H3K9me3_Setdb1KO_rep1

value: Mouse embryonic stem cells


title: EC_BW25113_CoIP_SpyCas9-3xF_native_leader_array_R1


title: B7


title: EC_BW25113_CoIP_SpyCas9-3xF_native_leader_array_R2


title: EC_BW25113_CoIP_SpyCas9_native_leader_array_R1


title: ChIP_H3K9me3_WT_rep2


title: ATO rep2

city: Shanghai

first: Ni

last: Yan

address: 197, Ruijin Er Street

country: China

institute: Shanghaijiaotong University

data_processing:
HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) used for basecalling.
FastQC (v0.10.1) for quality control. Using Cutadapt (v1.9.1) to remove adapter sequences and poor-quality reads. HISAT2 (v2.0.1) was used to aligned filtered-reads to the Mus musculus genome (GRCm38).
Gene-level read counts were generated from aligned reads by HTSeq (v0.6.1), and differential gene expression analysis was carried out using the R (v4.1.0) limma package (v 3.47.15).
Genome_build: GRCm38
Supplementary_files_format_and_content: The txt file includes FPKM values and counts for every gene and every sample.

extract_protocol:
RNA samples for sequencing were prepared using a total RNA purification kit (B518651, Sangon Biotech) from hippocampus samples. Sequencing libraries were constructed from 1 μg of purified mRNA using Illumina TruSeq RNA Sample Prep Kit (FC-122-1001, CA, USA).
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: tissue

value: Hippocampus

tag: age

value: 11-12 weeks of age

value: 5000 ppb ATO developmental exposure


title: ChIP_H3K9me3_WT_rep1


title: Bigelowiella_natans_KAS_rep1

city: Stanford

first: Georgi

middle: Kolev

last: Marinov

state: California

address: 279 Campus Drive West, Beckman Center, B-257A/259

department: Genetics

institute: STANFORD UNIVERSITY

data_processing:
Library strategy: KAS-Seq
read alignment, ATAC-seq and KAS-seq data: Bowtie 1.0.1
bigWig file generation: custom code
read alignment, Hi-C data: Juicer tools 1.8.9
Genome_build: Bigelowiella_natans_CCMP2755_v1.0 GCA_000320545.1
Supplementary_files_format_and_content: bedGraph of read coverage, normalized to RPM for bigWig files, .hic file for Hi-C

organism: Bigelowiella natans

growth_protocol: Bigelowiella natans strain CCMP2755 starting cultures were obtained from NCMA (National Center for Marine Algae and Microbiota) and cultured in L1-Si media on a 12-h-light:12-h-dark cycle.

extract_protocol:
B. natans cells were pelleted by centrifugation at 1000 g for 5 minutes at room temperature, then resuspended in 500 uL of media supplemented with 5 uM N3-kethoxal (final concentration). Cells were incubated at room temperature for 10 minutes, then centrifuged at 1000 g for 5 minutes at room temperature to remove the media with the kethoxal, and resuspended in 100 uL cold 1x PBS. Genomic DNA was then extracted using the Monarch gDNA Purification Kit (NEB T3010S) following the standard protocol but with elution using 85 uL 25 mM K3BO3 at pH 7.0.
The click reaction was carried out by combining 87.5 uL purified and sheared DNA, 2.5 uL 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma 760749), and 10 uL 10x PBS in a final volume of 100 uL. The reaction was incubated at 37C for 90 minutes. DNA was purified using AMPure XP beads (50 uL for a 100 uL reaction or 100 uL for a 200 uL reaction), beads were washed on a magnetic stand twice with 80% EtOH, and eluted in 130 uL 25mM K3BO3. Purified DNA was then sheared on a Covaris E220 instrument down to ~150-400 bp size. For streptavidin pulldown of biotin-labeled DNA, 10 uL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) were separated on a magnetic stand, then washed with 300 uL of 1x TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20). The beads were resuspended in 300 uL of 2x Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), the sonicated DNA was added (diluted to a final volume of 300 uL if necessary), and the beads were incubated for >=15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 300 uL of 1x TWB, and heated at 55C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55C incubation were repeated. Final libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645) as follows. End repair was carried out by resuspending beads in 50 uL 1x EB buffer, and adding 3 uL NEB Ultra End Repair Enzyme and 7 uL NEB Ultra End Repair Enzyme, followed by incubation at 20C for 30 minutes (in a Thermomixer, with shaking at 1,000 rpm) and then at 65C for 30 minutes.  Adapters were ligated to DNA fragments by adding 30 uL Blunt Ligation mix, 1 uL Ligation Enhancer and 2.5 uL NEB Adapter, incubating at 20C for 20 minutes, adding 3 uL USER enzyme, and incubating at 37C for 15 minutes (in a Thermomixer, with shaking at 1,000 rpm) . Beads were then separated on a magnetic stand, and washed with 300 uL TWB for 2 minutes at 55C, 1000 rpm in a Thermomixer. After separation on a magnetic stand, beads were washed in 100 uL 0.1 x TE buffer, then resuspended in 15 uL 0.1 x TE buffer, and heated at 98C for 10 minutes. For PCR, 5 uL of each of the i5 and i7 NEB Next sequencing adapters were added together with 25 uL 2x NEB Ultra PCR Mater Mix. PCR was carried out with a 98C incubation for 30 seconds and 12 cycles of 98C for 10 seconds, 65C for 30 seconds, and 72C for 1 minute, followed by incubation at 72C for 5 minutes. Beads were separated on a magnetic stand, and the supernatant was cleaned up using 1.8x AMPure XP beads. Libraries were sequenced in a paired-end format on a Illumina NextSeq instrument using NextSeq 500/550 high output kits (2x36 cycles).


title: C1


title: Bigelowiella_natans_ATAC_rep1

data_processing:
read alignment, ATAC-seq and KAS-seq data: Bowtie 1.0.1
bigWig file generation: custom code
read alignment, Hi-C data: Juicer tools 1.8.9
Genome_build: Bigelowiella_natans_CCMP2755_v1.0 GCA_000320545.1
Supplementary_files_format_and_content: bedGraph of read coverage, normalized to RPM for bigWig files, .hic file for Hi-C

extract_protocol:
ATAC-seq experiments were performed following the omniATAC protocol. Briefly, ~1M B. natans cells were centrifuged at 1,000 g, then resuspended in 500 uL 1x PBS and centrifuged again. Cells were then resuspended in 50 uL ATAC-RSB-Lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630, 0.1% Tween-20, 0.01% Digitonin) and incubated on ice for 3 minutes. Subsequently 1 mL ATAC-RSB-Wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.01% Digitonin) were added, the tubes were inverted several times, and nuclei were centrifuged at 500 g for 5 min at 4C. Transposition was carried out by resuspending nuclei in a mix of 25 uL 2x TD buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl2, 20% Dimethyl Formamide), 2.5 uL transposase (custom produced) and 22.5 uL nuclease-free H2O, and incubating at 37C for 30 min in a Thermomixer at 1000 RPM.
Transposed DNA was isolated using the MinElute PCR Purification Kit (Qiagen Cat# 28004/28006), and PCR amplified for 10 cycles using the usual ATAC-seq settings. Libraries were purified using the MinElute kit, then sequenced on a Illumina NextSeq 550 instrument as 2x36mers.


title: ChIP_SETDB1_WT_rep2

value: SETDB1, ProteinTech 11231-1-AP


title: Bigelowiella_natans_ATAC_rep2


title: Ctr rep2

value: Untreated


title: ATO rep1


title: Ctr rep1


title: Bigelowiella_natans_Hi-C

extract_protocol:
B. natans cells were first crosslinked using 37% formaldehyde (Sigma) at a final concentration of 1% for 15 minutes at room temperature. Formaldehyde was then quenched using 2.5 M Glycine at a final concentration of 0.25 M. Cells were subsequently centrifuged at 2,000 g for 5 minutes, washed once in 1x PBS, and stored at -80C.  Cell lysis was initiated by incubation with 250 uL of cold Hi-C Lysis Buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630) on ice for 15 minutes, followed by centrifugation at 2,500 g for 5 minutes, a wash with 500 uL of cold Hi-C Lysis Buffer, and centrifugation at 2,500 g for 5 minutes. The pellet was the resuspended in 50 uL of 0.5% SDS and incubated at 62C for 10 minutes. SDS was quenched by adding 145 uL of H$_2$O and 25 uL of 10% Triton X-100 and incubating at 37C for 15 minutes. Restriction digestion was carried out by adding 25 uL of 10x NEBuffer 2 and 100 U of the MluCI restriction enzyme (NEB, R0538) and incubating for >=2 hours at 37C in a Thermomixer at 900 rpm. The MluCI restriction enzyme was chosen as more suitable for the highly AT-rich nucleomorph genome. The reaction was then incubated at 62C for 20 minutes in order to inactivate the restriction enzyme. Fragment ends were filled in by adding 37.5 uL of 0.4 mM biotin-14-dATP (ThermoFisher Scientific, # 19524-016), 1.5 uL each of 10 mM dCTP, dGTP and dTTP, and 8 uL of 5U/uL DNA Polymerase I Large (Klenow) Fragment (NEB M0210). The reaction was the incubated at 37C in a Thermomixer at 900 rpm for 45 minutes. Fragment end ligation was carried out by adding 663 uL H$_2$O, 120 uL 10x NEB T4 DNA ligase buffer (NEB B0202), 100 uL of 10% Triton X-100, 12 uL of 10 mg/mL Bovine Serum Albumin (100x BSA, NEB), 5 uL of 400 U/uL T4 DNA Ligase (NEB M0202), and incubating at room temperature for $\geq$4 hours with rotation.  Nuclei were then pelleted by centrifugation at 2,000 g for 5 minutes; the pellet was resuspended in 200 uL ChIP Elution Buffer (1% SDS, 0.1 M NaHCO3), Proteinase K was added, and incubated at 65C overnight to reverse crosslinks.  After addition of 600 uL 1xTE buffer, DNA was sheared using a Covaris E220 instrument. DNA was then purified using the MinElute PCR Purificaiton Kit (Qiagen #28006), with elution in a total volume of 300 uL 1x EB buffer.
For streptavidin pulldown of biotin-labeled DNA, 150 uL of 10 mg/mL Dynabeads MyOne Streptavidin T1 beads (Life Technologies, 65602) were separated on a magnetic stand, then washed with 400 uL of 1x TWB (Tween Washing Buffer; 5 mM Tris-HCl pH 7.5; 0.5 mM EDTA; 1 M NaCl; 0.05% Tween 20). The beads were resuspended in 300 uL of 2x Binding Buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA; 2 M NaCl), the sonicated DNA was added, and the beads were incubated for >=15 minutes at room temperature on a rotator. After separation on a magnetic stand, the beads were washed with 600 uL of 1x TWB, and heated at 55C in a Thermomixer with shaking for 2 minutes. After removal of the supernatant on a magnetic stand, the TWB wash and 55C incubation were repeated.  Final libraries were prepared on beads using the NEBNext Ultra II DNA Library Prep Kit (NEB, #E7645) as follows. End repair was carried out by resuspending beads in 50 uL 1x EB buffer, and adding 3 uL NEB Ultra End Repair Enzyme and 7 uL NEB Ultra End Repair Enzyme, followed by incubation at 20C for 30 minutes and then at 65C for 30 minutes.  Adapters were ligated to DNA fragments by adding 30 uL Blunt Ligation mix, 1 uL Ligation Enhancer and 2.5 uL NEB Adapter, incubating at 20C for 20 minutes, adding 3 uL USER enzyme, and incubating at 37C for 15 minutes.  Beads were then separated on a magnetic stand, and washed with 600 uL TWB for 2 minutes at 55C, 1000 rpm in a Thermomixer. After separation on a magnetic stand, beads were washed in 100 uL 0.1 x TE buffer, then resuspended in 16 uL 0.1 x TE buffer, and heated at 98C for 10 minutes.  For PCR, 5 uL of each of the i5 and i7 NEB Next sequencing adapters were added together with 25 uL 2x NEB Ultra PCR Mater Mix. PCR was carried out with a 98C incubation for 30 seconds and 12 cycles of 98C for 10 seconds, 65C for 30 seconds, and 72C for 1 minute, followed by incubation at 72C for 5 minutes.  Beads were separated on a magnetic stand, and the supernatant was cleaned up using 1.8x AMPure XP beads.  Libraries were sequenced in a paired-end format on a Illumina NextSeq instrument using NextSeq 500/550 high output kits (either 2x75 or 2x36 cycles).


title: ChIP_SETDB1_WT_rep1


title: ChIP_Input


title: B1


title: A1


title: mTEClo/int OTII Input replicate

status: Public on Jan 24 2022

city: Nantes

first: Matthieu

last: Giraud

address: 30 bd Jean Monnet

department: CRTI

institute: INSERM _ Université de Nantes

data_processing:
Illumina HiSeq 2000 machine
paired-end 75bp reads
Alignment to the mm10 genome was done using Bowtie 2. Duplicate alignments were removed using samtools and the command ligne: "samtools view -S -hf 0x2 alignments.sam | grep -v "XS:i:" | foo.py > filtered.alignments.sam" the foo.py script been available online: "https://www.biostars.org/p/95929/".
Peak calling for a sample (in comparison to the inputs) was done using macs2 callpeak and the options -f BAMPE and --SPMR. The option --broad was used for H3K27me3
Enrichment for a sample compared to the inputs, was calculated using macs2 bdgcmp and the option -m FE
ceas was used to calculate the average entrichment at the TSS for H3K4me3 or within the gene body and its upstream and downstream regions for H3K27me3
Genome_build: mm10
Supplementary_files_format_and_content: wig and peak files were generated using macs2

source_name: medullary thymic epithelial cells

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Nano-ChIP-seq libraries were prepared with the TruSeq ChIP Sample Preparation Kit (Illumina).

treatment_protocol: mTEClo/int were isolated from individual mice using a BD FACS Aria III cell sorter, following the typical isolation procedure

value: thymus

value: CD80 lo/int medullary epithelial cells

tag: genotype

value: OTII


title: mTEClo/int RipmOVA Input replicate

value: RipmOVAxOTII


title: mTEClo/int RipmOVA Input


title: Bigelowiella_natans_KAS_rep2


title: mTEClo/int WT Input replicate

value: WT


title: A7

value: Growth Control


title: mTEClo/int WT H3K27me3

value: H3K27me3 (Cell Signaling Technology, ref:9733)


title: mTEClo/int WT Input


title: mTEClo/int WT H3K4me3

value: H3K4me3 (Abcam, ref:ab8580)


title: mTEClo/int OTII Input


title: mTEClo/int RipmOVA H3K27me3


title: mTEClo/int RipmOVA H3K4me3


title: mTEClo/int OTII H3K27me3


title: mTEClo/int OTII H3K4me3


title: D4 PB Sample 1

city: ROCHESTER

first: Diane

last: Jelinek

state: MN

address: 200 1st St SW

institute: Mayo Clinic

data_processing:
Base-calling was performed using Illumina’s RTA version 2.7.7.
Sequencing reads were analyzed using the RNA-seq bioinformatics pipeline MAP-RSeq
Data were normalized using the Trimmed Mean of M-values (TMM)
Genome_build: hg18
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

source_name: Peripheral Blood B cells

growth_protocol: Peripheral blood B cells were driven to differentiation into plasma cells with various cytokine cocktails

extract_protocol:
RNA prepared by RNeasy Micro Kit
RNA libraries were prepared using 100 ng of high quality total RNA according to the manufacturer’s instructions for the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA) employing poly-A mRNA enrichment using oligo dT magnetic beads.

tag: differentiation stage

value: D4 Plasmablasts

value: Peripheral Blood B cells


title: D0 PB Sample 1

value: D0 resting B cells


title: D4 PB Sample 2


title: D0 PB Sample 3


title: D10 PB Sample 3

value: D10 IVPCs


title: D4 PB Sample 3


title: D10 PB Sample 1


title: D10 PB Sample 2


title: D0 PB Sample 2


title: EtOH_A12_4Cseq, GBS4, rep3

city: Research Triangle Park

first: Jackson

middle: Andrew

last: Hoffman

state: North Carolina

address: 111 TW Alexander Dr, Building 101 Room C432

department: DIR

institute: NIEHS

data_processing:
Library strategy: 4C-seq
Adapter sequences were trimmed from ChIP-seq reads using Cutadapt (Martin, 2011) and low quality reads were removed from analysis using Sickle (Joshi NA et al., 2011). Alignment was performed with Bowtie2 (Langmead and Salzberg, 2012). Aligned reads were sorted and processed with Samtools (Li et al., 2009) and de-duplicated using Picard Tools (http://broadinstitute.github.io/picard). Peaks were called using MACS2 (Zhang et al., 2008) and Homer (Heinz et al., 2010) using a false discovery rate cutoff of 0.001, and regions of high depth or with high signal in untreated or input samples were used to filter out false positive peak calls. Peak overlaps and distance analyses were performed using Bedtools (Quinlan and Hall, 2010). Coverage files and heatmaps were generated using Deeptools (Ramírez et al., 2016).
For 4C-seq, data was processed using Pipe4C (Krijger et al., 2020) and run via Rscript for R version 3.6.1 with the following parameters: trimLength=0, mismatchMax=0, and minAmountReads=10. Data QC involved reviewing output reports to verify that the overall alignment rate was typically >85% and resulted in at least 1 million reads aligned to the fragment index. Results were visualized using ggplot2 (Wickham, 2016).
Genome_build: hg19
Supplementary_files_format_and_content: RDS

source_name: A1-2 breast cancer cells

growth_protocol: Cells were cultured in modified Eagle medium (MEM) containing 10% fetal bovine serum, 1X Penicillin/Streptomycin (Sigma P0781), 1% HEPES (Sigma H0887), 1X Glutamax (Gibco 35050), and 250 ug/ml G418 (Gibco 10131).

extract_protocol:
For ChIPseq, cells were fixed in PBS with 1% methanol-free formaldehyde (Thermoscientific 28906) at 37C for 10 min and quenched with glycine. Cell pellets were resuspended in MNase swelling buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 5 mM Sodium Butyrate, 0.5 mM PMSF, and protease inhibitor cocktail (PIC) (ThermoFisher Scientific, 78429)) for enzymatic fragmentation or into hypotonic buffer (10 mM HEPES-NaOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10% glycerol, 0.1% Triton X-100, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) for mechanical sonication. Cells were incubated on ice for 10 min then subjected to Dounce homogenization using 20-strokes (Duran Wheaton Kimble, 357542).  For enzymatic fragmentation with MNase, nuclei were sedimented through digestion buffer (15mM Hepes pH 7.9, 60mM KCl, 15mM NaCl, 0.32M Sucrose, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) by centrifugation for 7 min at 930g (4C). Nuclei pellets were fully resuspended in digestion buffer containing 3.3 mM CaCl2 and digested with MNase (0.7U/1x10^6 cells, Worthington, cat#) at 37C for 15min. MNase fragmentation was stopped by addition of 10 mM EGTA and incubation for 5 min on ice. Digested nuclei were lysed in LB3 buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N- Lauroylsarcosine, 1% Triton X-100, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) using 5-passages through a 25g ( 0.508 mm x 1.6 cm) needle and brief sonication using 4 cycles (30 sec on/off, power high, 4C) with a Bioruptor sonicator (source). MNase digested chromatin lysate was recovered after 15 min centrifugation at 13,000g (4C).  For mechanical sonication, nuclei pellets were resuspended in Shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 5 mM Sodium Butyrate, 0.1% SDS, and PIC) and chromatin was fragmented by sonication using the Covaris S220 (Peak Power, 175; Duty Factor, 10; Cycles/Burst, 200; Total Time, 600 sec). Sonicated chromatin was recovered by centrifugation at 20,000g (4C) for 10 min.
4C-seq was performed according to the published protocol (Krijger et al., 2020). Briefly, batches of 5-10 million cells were fixed in 2% Formaldehyde solution and flash frozen in liquid nitrogen. A single pellet of 5-10 million cells was used for each 4C replicate. Chromatin was first digested with NlaIII, ligated with T4 DNA Ligase to generate the 3C template, and purified with NucleoMag PCR beads. The purified 3C template was subsequently digested with DpnII, ligated with T4 DNA Ligase to generate the 4C template, and again purified with NucleoMag PCR beads.
ChIP-seq libraries were generated using the ACCEL-NGS® 2S PLUS DNA library kit according to manufacturer’s protocol and sequenced on the Illumina NextSeq platform.
4C-seq libraries were generated by two-step PCR using Expand Long Template PCR kit. The first step PCR reaction used reading and non-reading primers to amplify 4C fragments and was purified with a 0.8X AMPure XP purification. The second step PCR reaction added sequencing adapters and indexes and was purified using Qiagen PCR purification kits. Libraries were sequenced on an Illumina MiSeq with a target of 1-2 million single-end, 100bp reads per library.

treatment_protocol: For Dexamethasone treatments, cells were initially cultured for 24 hours in reduced-serum, hormone-stripped media (Phenol-red free MEM (Gibco 51200) with 5% Charcoal/Dextran treated FBS (R&D Systems S11650), 1X Penicillin/Streptomycin (Sigma P0781), 1% HEPES (Sigma H0887), 1X Glutamax (Gibco 35050), and 250 ug/ml G418 (Gibco 10131)). Subsequently, Dexamethasone (Sigma D4902) was added to the media at 100 nM for all treatments timepoints and 9.5 x 10-4% ethanol was used as vehicle control.

value: A1-2 breast cancer cells

value: EtOH

tag: 4c viewpoint

value: DDIT4 GBS4


title: Dex1hr_A12_4Cseq, 22kb, rep2

value: 1hr Dex

value: DDIT4 -22kb


title: OML1-A40#2

type: genomic

city: Chiayi County

first: Jie Ting

last: Low

state: Minxiong Township

address: No. 168, Daxue Road, Section 1

country: Taiwan

institute: National Chung Cheng University

description:
genomic DNA from OML1R
tumor cell

hyb_protocol: bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation27 Beadchip using standard Illumina protocol

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting

data_processing: GenomeStudio

label: Cy3 and Cy5

label_protocol: Standard Illumina Protocol

growth_protocol: OML1 and OML1R cells were grown in RPMI medium supplemented with 10% FBS, and  1% penicillin-streptomycin. Cells were cultured at 37°C and 5% CO2 incubator.

extract_protocol: genomic DNA was  purified from cell extract using genomic DNA mini kit  Kit (Geneaid, GT100, Taiwan) according to standard instructions.Bisulfite conversion of genomic DNA was performed with EZ DNA methylation Kit (Zymo Research,  Orange, CA, #D5002) following the manufacturer's protocol with modifications for the Infinium Methylation Assay.

treatment_protocol: We constructed OML1-R from parental OML1 cells by using fractionated irradiations. A fraction size of 5 Gy was delivered per 5-7 days. By eight fractions, a total of 40 Gy were delivered on parental OML1 cells to construct OML1-R cells.

value: Buccal mucosa squamous carcinoma


title: Dex1hr_A12_4Cseq, GBS4, rep3


title: Si-METTL3.input

city: wuxi

first: Siyu

last: Liu

address: Street of River Deans, Binhu District, Wuxi City

institute: Jiangnan University Affiliated Hospital

data_processing:
m6A: Briefly, Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3). First, clean reads of all libraries were aligned to the reference genome (HG19)  by Hisat2 software (v2.0.4).
m6A: Methylated sites on RNAs (peaks) were identified by MACS software. Differentially methylated sites were identified by diffReps. These peaks identified by both softwares overlapping with exons of mRNA were figured out and chosen by home-made scripts. GO and Pathway enrichment analysis were performed by the differentially methylated protein coding genes.
input: Paired-end reads were harvested from Illumina HiSeq 4000 sequencer, and were quality controlled by Q30. After 3’ adaptor-trimming and low quality reads removing by cutadapt software (v1.9.3), the high quality clean reads were aligned to the reference genome (UCSC hg19) with hisat2 software (v2.0.4).
input: Then, guided by the Ensembl gtf gene annotation file, cuffdiff software (part of cufflinks) was used to get the gene level FPKM as the expression profiles of mRNA, and fold change and p-value were calculated based on FPKM, differentially expressed mRNA were identified. GO and Pathway enrichment analysis were performed based on the differentially expressed mRNAs.
Genome_build: UCSC HG19
Supplementary_files_format_and_content: excel format

source_name: skin

extract_protocol:
Total RNA was isolated using trizol.Total RNA (1 μg) was used for removing the rRNAs using NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA)  following the manufacturer's instructions
m6a: m6A RNA-Seq service was provided by Cloudseq Biotech Inc. (Shanghai, China). Briefly, m6A RNA immunoprecipitation was performed with the GenSeq™ m6A-MeRIP Kit (GenSeq Inc., China) by following the manufacturer's instructions. Both the input samples without immunoprecipitation and the m6A IP samples were used for RNA-seq library generation with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA).
m6a:The library quality was evaluated with BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150bp paired-end reads.
input: RNA high throughput sequencing was performed by Cloud-Seq Biotech (Shanghai, China). Briefly, total RNA was used for removing the rRNAs with NEBNext rRNA Depletion Kit (New England Biolabs, Inc., Massachusetts, USA) following the manufacturer's instructions. RNA libraries were constructed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, Inc., Massachusetts,USA) according to the manufacturer’s instructions.
input: Libraries were controlled for quality and quantified using the BioAnalyzer 2100 system (Agilent Technologies, Inc., USA). Library sequencing was performed on an illumina Hiseq instrument with 150bp paired end reads.

value: skin

tag: disease state

value: hypertrophic scar

tag: knockdown

tag: rip antibody

value: none (input)


title: Si-METTL3.IP


title: Dex1hr_A12_4Cseq, 22kb, rep3

tag: cell line

tag: treatment


title: EtOH_A12_4Cseq, 22kb, rep3


title: OML1-A0#1

description:
genomic DNA from OML1
tumor cell

tag: cell type


title: EtOH_A12_4Cseq, 22kb, rep2


title: Dex1hr_A12_4Cseq, GBS4, rep2


title: Si-NC.input

description: Si-NC.input

value: si-NC


title: OML1-A40#1

description:
genomic DNA from OML1R
tumor cell


title: EtOH_A12_4Cseq, GBS4, rep2


title: Si-NC.IP

description: Si-NC.IP


title: Empty vector FLAG IP rep 2 input

city: La Jolla

first: Gene

last: Yeo

state: CA

address: 2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab

institute: UCSD

description: 4P_IP_vs_11P_input_IDR_6P_IP_vs_12P_input.bed

data_processing:
Sequenced reads were reformatted to include randomers in read headers with umi_tools (1.0.0). Args: --random-seed 1 --bc-pattern NNNNNNNNNN
Reads were then trimmed with cutadapt (1.14). Args: --match-read-wildcards -O 1 --times 1 -e 0.1 --quality-cutoff 6 -m 18 -a InvRNA*.fasta (fasta sequences can be found at: https://github.com/YeoLab/eclip/tree/master/example/inputs/)
Reads were then trimmed once more with cutadapt (1.14) to remove double-ligation events. Args: --match-read-wildcards -O 5 --times 1 -e 0.1 --quality-cutoff 6 -m 18  -a InvRNA*.fasta (fasta sequences can be found at: https://github.com/YeoLab/eclip/tree/master/example/inputs/)
Trimmed reads were then mapped with STAR (2.4.0i) against a human-specific repeat element database (RepBase 18.05). Args: --runThreadN 16 \  --genomeDir human_repbase \  --readFilesIn path/to/read1 \  --outFileNamePrefix out_prefix \  --outReadsUnmapped Fastx \  --outSAMtype BAM Unsorted \  --outSAMattributes All \  --outSAMunmapped Within \  --outSAMattrRGline ID:foo \  --outFilterType BySJout \  --outFilterMultimapNmax 30 \  --outFilterMultimapScoreRange 1 \  --outFilterScoreMin 10 \  --alignEndsType EndToEnd
Unmapped reads filtered of repeat elements were then mapped with STAR (2.4.0i) against a human genome (GRCh38). Args: --runThreadN 16 \  --genomeDir genomedir \  --readFilesIn /path/to/read1 \  --outFileNamePrefix out_prefix \  --outReadsUnmapped Fastx \  --outSAMtype BAM   Unsorted \  --outSAMattributes All \  --outSAMunmapped Within \  --outSAMattrRGline ID:foo \  --outFilterType BySJout \  --outFilterMultimapNmax 1 \  --outFilterMultimapScoreRange 1 \  --outFilterScoreMin 10 \  --alignEndsType EndToEnd
Aligned reads were sorted with samtools (1.6)
Sorted reads were collapsed with umi_tools (1.0.0). Args: --random-seed 1 --method unique
BAM files were used to identify peak clusters with Clipper (1.2.2). Args: --species GRCh38 --bam path/to/input.bam --timeout 3600000 --maxgenes 1000000 --save-pickle --outfile path/to/output.bam
Peak clusters were normalized using BAM files for IP against BAM files for INPUT with peaksnormalize.pl (overlap_peakfi_with_bam_PE.pl), included in eclip 0.1.5+.
Overlapping normalized peak regions were merged with compress_l2foldenrpeakfi_for_replicate_overlapping_bedformat.pl, included within eclip-0.1.5+
Normalized peak (compressed.bed) files were ranked by entropy score (make_informationcontent_from_peaks.pl included within the merge_peaks pipeline) and used as inputs to IDR (2.0.2) to determine reproducible peaks.
Genome_build: GRCh38

growth_protocol: HEK293 cells were maintained in DMEM (4.5 g/L D-glucose) supplemented with 10% FBS (Gibco) at 37o C with 5% CO2. Cells were periodically passaged once at 70-90% confluency by dissociating with TrypLE Express Enzyme (Gibco) at a ratio of 1:10

extract_protocol:
Biological replicates of confluent 10cm plates of 293T cells were treated with 400mJ/cm2 of UV twice using the Stratalinker 2400, harvested in ice cold PBS and pellets flash frozen in liquid nitrogen and stored in -80 degrees until ready to IP in a subsequent protocol exactly as detailed in Van Nostrand et al (2016), cutting the nitrocellulose membrane from 115 kDa and up. Sequencing was performed on Illumina HiSeq 4000 with paired end reads and data processed through Dr. Yeo's eCLIP pipeline.
Libraries were constructed as per the eCLIP protocol using Illumina 50x forward and 70x reverse primers.

treatment_protocol: HEK293 cells were UV cross-linked with 400mJ/cm2 twice and processed through the eCLIP protocol as previously described (van Nostrand et al, 2016).

tag: antibody manufacturer

value: Sigma-Aldrich

tag: antibody catalog #

value: Monoclonal ANTI-FLAG M2 antibody (F1804)

tag: cell manufacturer

value: Takara Bio

value: Lenti-X 293T (HEK 293T)

tag: adapter


title: OML1-A0#2

description:
genomic DNA from OML1
tumor cell


title: Dex1hr_A12_4Cseq, 22kb, rep1


title: EtOH_A12_4Cseq, 22kb, rep1


title: Empty vector FLAG IP rep 1 input


title: Dex1hr_A12_4Cseq, GBS4, rep1


title: 3xFLAG-dSpCas9 rep 2 FLAG IP

description:
4P_IP_vs_4N_input_IDR_6P_IP_vs_6N_input.bed
4P_IP_vs_11P_input_IDR_6P_IP_vs_12P_input.bed


title: EtOH_A12_4Cseq, GBS4, rep1


title: 3xFLAG-dSpCas9 rep 2 size-matched input

description: 4P_IP_vs_4N_input_IDR_6P_IP_vs_6N_input.bed


title: Dex1hr_GBS4eKO_4Cseq [GBS4eKO_Dex_4C.rep2]

data_processing:
library strategy: 4C-seq
Adapter sequences were trimmed from ChIP-seq reads using Cutadapt (Martin, 2011) and low quality reads were removed from analysis using Sickle (Joshi NA et al., 2011). Alignment was performed with Bowtie2 (Langmead and Salzberg, 2012). Aligned reads were sorted and processed with Samtools (Li et al., 2009) and de-duplicated using Picard Tools (http://broadinstitute.github.io/picard). Peaks were called using MACS2 (Zhang et al., 2008) and Homer (Heinz et al., 2010) using a false discovery rate cutoff of 0.001, and regions of high depth or with high signal in untreated or input samples were used to filter out false positive peak calls. Peak overlaps and distance analyses were performed using Bedtools (Quinlan and Hall, 2010). Coverage files and heatmaps were generated using Deeptools (Ramírez et al., 2016).
For 4C-seq, data was processed using Pipe4C (Krijger et al., 2020) and run via Rscript for R version 3.6.1 with the following parameters: trimLength=0, mismatchMax=0, and minAmountReads=10. Data QC involved reviewing output reports to verify that the overall alignment rate was typically >85% and resulted in at least 1 million reads aligned to the fragment index. Results were visualized using ggplot2 (Wickham, 2016).
Genome_build: hg19
Supplementary_files_format_and_content: bw

extract_protocol:
For ChIPseq, cells were fixed in PBS with 1% methanol-free formaldehyde (Thermoscientific 28906) at 37C for 10 min and quenched with glycine. Cell pellets were resuspended in MNase swelling buffer (25 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 5 mM Sodium Butyrate, 0.5 mM PMSF, and protease inhibitor cocktail (PIC) (ThermoFisher Scientific, 78429)) for enzymatic fragmentation or into hypotonic buffer (10 mM HEPES-NaOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 340 mM sucrose, 10% glycerol, 0.1% Triton X-100, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) for mechanical sonication. Cells were incubated on ice for 10 min then subjected to Dounce homogenization using 20-strokes (Duran Wheaton Kimble, 357542). For enzymatic fragmentation with MNase, nuclei were sedimented through digestion buffer (15mM Hepes pH 7.9, 60mM KCl, 15mM NaCl, 0.32M Sucrose, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) by centrifugation for 7 min at 930g (4C). Nuclei pellets were fully resuspended in digestion buffer containing 3.3 mM CaCl2 and digested with MNase (0.7U/1x10^6 cells, Worthington, cat#) at 37C for 15min. MNase fragmentation was stopped by addition of 10 mM EGTA and incubation for 5 min on ice. Digested nuclei were lysed in LB3 buffer (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N- Lauroylsarcosine, 1% Triton X-100, 5 mM Sodium Butyrate, 0.5 mM PMSF, and PIC) using 5-passages through a 25g ( 0.508 mm x 1.6 cm) needle and brief sonication using 4 cycles (30 sec on/off, power high, 4C) with a Bioruptor sonicator (source). MNase digested chromatin lysate was recovered after 15 min centrifugation at 13,000g (4C).  For mechanical sonication, nuclei pellets were resuspended in Shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 5 mM Sodium Butyrate, 0.1% SDS, and PIC) and chromatin was fragmented by sonication using the Covaris S220 (Peak Power, 175; Duty Factor, 10; Cycles/Burst, 200; Total Time, 600 sec). Sonicated chromatin was recovered by centrifugation at 20,000g (4C) for 10 min.
4C-seq was performed according to the published protocol (Krijger et al., 2020). Briefly, batches of 5-10 million cells were fixed in 2% Formaldehyde solution and flash frozen in liquid nitrogen. A single pellet of 5-10 million cells was used for each 4C replicate. Chromatin was first digested with NlaIII, ligated with T4 DNA Ligase to generate the 3C template, and purified with NucleoMag PCR beads. The purified 3C template was subsequently digested with DpnII, ligated with T4 DNA Ligase to generate the 4C template, and again purified with NucleoMag PCR beads.
ChIP-seq libraries were generated using the ACCEL-NGS® 2S PLUS DNA library kit according to manufacturer’s protocol and sequenced on the Illumina NextSeq platform.
4C-seq libraries were generated by two-step PCR using Expand Long Template PCR kit. The first step PCR reaction used reading and non-reading primers to amplify 4C fragments and was purified with a 0.8X AMPure XP purification. The second step PCR reaction added sequencing adapters and indexes and was purified using Qiagen PCR purification kits. Libraries were sequenced on an Illumina MiSeq with a target of 1-2 million single-end, 100bp reads per library.

tag: cell line/type

value: A1-2 GBS4eKO breast cancer cells

value: DDIT4 TSS


title: 3xFLAG-dSpCas9 rep 1 size-matched input


title: Dex1hr_GBS4eKO_4Cseq [GBS4eKO_Dex_4C.rep3]


title: Empty vector V5 IP rep 1 input

value: ThermoFisher Scientific

value: V5 Tag Monoclonal antibody (R960-25)


title: Empty vector V5 IP rep 2 input


title: V5-dSpCas9 rep 2 size-matched input


title: EtOH_GBS4eKO_4Cseq [GBS4eKO_EtOH_4C.rep3]

value: EtOH


title: Dex1hr_GBS4eKO_4Cseq [GBS4eKO_Dex_4C.rep1]


title: EtOH_GBS4eKO_4Cseq [GBS4eKO_EtOH_4C.rep2]


title: V5-dSpCas9 rep 1 V5 IP

description:
1P_vs_1N.vs.2P_vs_2N.bed
1P_vs_8P.vs.2P_vs_9P.bed


title: 3xFLAG-dSpCas9 rep 1 FLAG IP


title: V5-dSpCas9 rep 2 V5 IP


title: CWRR1 SOX2-KO #2

city: Chicago

first: Donald

last: Vander Griend

state: IL

address: 909 S. Wolcott Ave, COMRB Room 5051

department: Pathology

institute: The University of Illinois at Chicago

data_processing:
All RNA-seq reads were analyzed using the FastQC tool suite
Low quality and adapter sequences were trimmed using trimmomatic
The trimmed reads were then aligned to the reference genome GRCh38 using STAR (star) and the resulting alignments were sorted and converted to BAM format using Samtools.
Duplicate reads were subsequently removed with Picard tools
Basecalling was performed using Illumina CASAVA version 1.8.
Adapter sequences and low quality reads were trimmed using Trimmomatic v0.38.
All reads were pseudo-aligned to the human transcriptome built from ENSEMBL Human GRCh38.p12 cDNA and ncRNA using kallisto (v0.43.1) with default settings for single-end reads with fragment length of 180 and standard deviation of 20.
Estimated transcript counts were summarized to the gene level using tximport (v1.8.0).
Filtering of lowly-expressed genes (<1 count per million in 6 of the 12 libraries), library normalization, and differential expression analysis was carried out in edgeR (v3.22.2) using the glmTreat method with a log fold change threshold of log2(1.5) and FDR < 0.05.
Genome_build: hg38 transcriptome
Supplementary_files_format_and_content: All processed data files are Tab-delimited text files with the final count per million (cpm) value from edgeR for every gene in each of the replicates for the condition specified by the filename.
Supplementary_files_format_and_content: TPM values for all 12 samples for all ENSEMBL genes in a matrtix

source_name: prostate

extract_protocol: none provided by the submitter

value: CWR-R1 cell line

value: castration-resistant

value: SOX2 negative


title: EtOH_GBS4eKO_4Cseq [GBS4eKO_EtOH_4C.rep1]


title: V5-dSpCas9 rep 1 size-matched input


title: CWRR1 SOX2-KO #1


title: Dex1hr_GBS3eKO_4Cseq [GBS3eKO_Dex_4C.rep1]

value: A1-2 GBS3eKO breast cancer cells

value: 1hr Dex


title: CWRR1 SOX2-KO #3


title: Dex1hr_GBS3eKO_4Cseq [GBS3eKO_Dex_4C.rep3]


title: CWRR1 Untreated #3

value: SOX2 positive


title: EtOH_GBS3eKO_4Cseq [GBS3eKO_EtOH_4C.rep2]


title: CWRR1 Untreated #2


title: iDART_10xGenomics_scDART-seq_APOBEC1-YTHmut Replicate 2

extract_protocol:
SMART-seq2 cells: Single cells were sorted using a FACS Aria II (BD) directly into lysis buffer provided with the SMART-seq HT Kit (Takara, Cat. 634438). The SMART-seq HT Kit was then used for reverse transcription and cDNA amplification. Sequencing libraries were constructed from cDNA using the Nextera XT kit (Illumina, FC-131-1096) and dual Nextera-compatible CDI indexes from IDT. Samples were pooled and sequenced targeting 5,000,000 reads/library (150bp paried-end) on a Novaseq6000 using the S4 flowcell.   10X Genomics cells: A single cell suspension was prepared according to manufacturer's instruction for Chromium v3 3' end sequencing (10x Genomics, Manual Part #CG00053). Samples were sequenced targeting 80,000 reads per cell on a Novaseq6000 using the S1 flowcell.  Bulk RNA-seq: Trizol was used to extract total RNA from iDART cells. The UltraII Directional RNA Library Prep Kit (NEB, E7760) with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, 7490) was used to generate sequencing libraries using 900ng of input RNA. The samples were sequenced targeting ~30 million reads (50bp paired-end sequencing) per sample on a Novaseq6000 using the S1 flowcell.
SMART-seq2 scRNA-seq: cDNA libraries were generated using the SMART-seq HT Kit (Takara, Cat. 634438). Final libraries were prepared using Nextera XT DNA Library Prep Kit (Illumina, FC-131-1096).   10x scRNA-seq: Libraries were generated from a single cell suspension using the 10x Genomics NextGEM Single Cell 3' Kit, v3.1 chemistry (10x Genomics)  Bulk-RNA seq: The UltraII Directional RNA Library Prep Kit (NEB, E7760) with NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, 7490) was used to generate sequencing libraries using 900ng of input RNA.

value: HEK293T Cells

tag: transgene

value: 1microg/mL doxycycline; 24 hours


title: EtOH_GBS3eKO_4Cseq [GBS3eKO_EtOH_4C.rep1]


title: WA01-Input, ChiP-Seq

description: input chromatin that was used as control for calling peaks against

data_processing:
Basecalling was performed using Illumina CASAVA version 1.8.
Adapter sequences and low quality reads were trimmed using Trimmomatic v0.38.
Duplicate reads were marked and removed using Picard Tools (v2.18.10).
Processed reads were then aligned to human reference genome hg38 (ENSEMBL release 93, GRCh38.p12) using Bowtie2 with default settings. Samtools was then used to change .sam files to .bam files, sort, and index the .bam files.
Peaks were called against sequenced input chromatin using MACS2 and pooling immunoprecipitated samples for either CWR-R1 or WA01 cells. Called peaks were then annotated to hg38 using the annotatePeaks.pl command in HOMER (v4.9.1).
Genome_build: hg38
Supplementary_files_format_and_content: Peak file with quantitative data for SOX2 peaks called in the CWR-R1 and WA01 cell line vs input DNA.; annotated_CWRR1-SOX2 binding, ChIP-Seq.txt and WA01-SOX2 binding, ChIP-Seq.txt: HOMER annotation of SOX2 peaks called in CWR-R1 and WA01cells, annotated to hg38.

source_name: human embryonic cell line

growth_protocol: CWRR1 cells were grown in RPMI medium containing 10% FBS, and supplemented with penicillin (50 units/ml), streptomycin (100 mg/ml), and puromycin (1 ug/mL).

extract_protocol:
Chromatin was harvested, sonicated, and immunoprecipitated (or not immunoprecipitated in the case of input DNA control) according to manufacturer protocol for Diagenode iDeal ChIP-seq kit for Transcription Factors (Cat# C01010055). Briefly, 25 million cells per replicate were fixed with formaldehyde at room temp for 15 mins before stopping fixation with glycine. Cells were then lysed and chromatin was harvestd and sonicated with a Diagenode Biorupter Pico for 5 cycles of 30 seconds on, 30 seconds off. Sheared chromatin size was checked with agarose gel electrophoresis and shown to be ~200 bp. MEIS1 bound chromatin was immunoprecipitated with MEIS1 ChIP-grade antibody (Cat# ab19867) at 4C overnight before being washed, eluted, and quantified with high sensitivity Qubit. IPs were performed in triplicate for LV-MEIS1 condition and in duplicate for HOXB13ko-LV-MEIS1 condition. Effective pulldown of MEIS1 was validated by western blot before library construction.
5ng of ChIP'd DNA (or of input DNA) was used as starting material for library construction using the KAPA LTP Library prep kit (Roche Cat# 07961863001) following manufacturer protocol for chromatin sheared to 175 bp. Briefly, end repair was performed on fragmented DNA, followed by A-tailing and Illumina TruSeq adapter ligation. Each library was then amplified with 13 cycles of PCR. Final library quality was assessed on an Agilent TapeStation 2200. Libraries were quantified using KAPA Universal Library Quantification Kit (Roche Cat# 07960140001) before pooling libraries in equamolar concentrations. Sequencing was performed on Illumina HiSeq 4000 using single end, 50bp reads.

treatment_protocol: All lines were seeded at 5 x 10^6 cells per dish in 15 cm dishes and were incubated at 37°C in 5% CO2 for 48 hrs prior to fixing and harvesting chromatin.

value: human embroynic cell line, pluripotent, and obtained from WiCell

tag: antibody used for chip

value: goat IgG


title: Dex1hr_GBS3eKO_4Cseq [GBS3eKO_Dex_4C.rep2]


title: EtOH_GBS3eKO_4Cseq [GBS3eKO_EtOH_4C.rep3]


title: DVG-WA01, SOX2-ChIP-Seq

value: Exogenous expression of SOX2

value: goat anti-SOX2 mAb (P48431, R&D Systems)


title: EtOH_GBS2eKO_4Cseq [GBS2eKO_EtOH_4C.rep3]

value: A1-2 GBS2eKO breast cancer cells


title: Dex1hr_GBS2eKO_4Cseq [GBS2eKO_Dex_4C.rep2]

value: DDIT4 TSS


title: Dex1hr_GBS2eKO_4Cseq [GBS2eKO_Dex_4C.rep3]


title: Dex1hr_GBS2eKO_4Cseq [GBS2eKO_Dex_4C.rep1]


title: HS_4SU.mRNA-seq_rep1

city: London

first: Richard

middle: James

last: Mitter

state: LONDON

address: 1 Midland Road

department: Bioinformatics & Biostatistics

institute: The Francis Crick Institute

description:
mRNA-Seq
poly(A)+ mRNA

data_processing:
cutadapt v1.18 was used to trim Illumina adapter sequence from all reads using the following settings: -a AGATCGGAAGAGC -e 0.1 -q 10 -m 25 -O 1
Trimmed reads were aligned against human genome version GRCh38 using STAR v2.5.2a with Ensembl release 86 transcript annotations.
Genome_build: GRCh38
Supplementary_files_format_and_content: Read depth coverage in bigWig format. Data are unscaled and represent reads mapping to both strands.

source_name: MRC5-VA cell line

growth_protocol: DMEM (Thermo Fisher Scientific, 11965118) supplemented with 10% v/v FBS (Clontech, 631106), 100 U/ml penicillin, 100 μg/ml streptomycin.

extract_protocol:
RNA was extracted using the RNeasy kit (Qiagen, 74104) according to the manufacturer’s instruction including the on-column DNase treatment. 4SU-residues biotinylation of 100 μg mammalian 4SU labelled RNA was carried out in a total volume of 250 μl, containing 10 mM Tris-HCl [pH 7.4], 1 mM EDTA and 5mg MTSEA biotin-XX linker (Biotium, BT90066) for 1 hour at room temperature in the dark. RNA was then purified by phenol:chloroform extraction, denatured by 10 min incubation at 65°C and added to 200 μL μMACS Streptavidin MicroBeads (Miltenyi, 130-074-101). RNA was incubated with beads for 15 min at room temperature and beads applied to a μColumn in the magnetic field of a μMACS magnetic separator. Beads were washed twice with pull-out wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). 4SU-RNA was eluted twice by addition of 100 mM DTT and RNA cleaned up using the RNeasy MinElute kit (Qiagen, 74204). 200ng total RNA was used for oligo(dT) purification.
Libraries were prepared using the KAPA mRNA HyperPrep kit (Roche).

treatment_protocol: Following a pre-treatment with HS for 1h and 45 min, RNA was labelled in vivo with 0.7mM 4SU for 90 min in HS conditions and collected for RNA extraction.

tag: condition

value: Heat shock


title: HS_4SU.mRNA-seq_rep2

description:
mRNA-Seq
poly(A)+ mRNA


title: ctrl_4SU.mRNA-seq_rep2

description:
mRNA-Seq
poly(A)+ mRNA

value: Control


title: Dex1hr_GBS1eKO_4Cseq [GBS1eKO_Dex_4C.rep3]

value: A1-2 GBS1eKO breast cancer cells


title: HS_FP.TTchem-seq_40min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

extract_protocol:
RNA was extracted by TRIzol (Thermo Fisher Scientific, 15596026) according to the manufacturer’s instructions. As a control for equal sample preparation, we spiked-in S. cerevisiae (strain BY4741, MATa, his3D1, leu2D0, met15D0, ura3D0) 4-thiouracil (4TU)-labelled RNA. S. cerevisiae were grown in YPD medium overnight, diluted to an OD600 of 0.1, and grown to mid-log phase (OD600 of 0.8) and labelled with 5 mM 4TU (Sigma-Aldrich, 440736) for 6 min. Total RNA was extracted using the PureLink RNA Mini kit (Thermo Fisher Scientific, 12183020) following the enzymatic protocol. For purification of 4SU labelled RNA, 100 μg mammalian 4SU labelled RNA was spiked-in 1/100 of 4TU-labelled S. cerevisiae RNA. The 101 μg RNA was fragmented by addition of 20 μL 1 M NaOH to fragment RNA and left on ice for 20 min. Fragmentation was stopped by addition of 80 μL 1 M Tris pH 6.8 and cleaned up twice with Micro Bio-Spin™ P-30 Gel Columns (BioRad, 7326223) according to the manufacturer’s instructions. Biotinylation of 4SU-residues were carried out in a total volume of 250 μl, containing 10 mM Tris-HCl [pH 7.4], 1 mM EDTA and 5mg MTSEA biotin-XX linker (Biotium, BT90066) for 30 min at room temperature in the dark. RNA was then purified by phenol:chloroform extraction, denatured by 10 min incubation at 65°C and added to 200 μL μMACS Streptavidin MicroBeads (Miltenyi, 130-074-101). RNA was incubated with beads for 15 min at room temperature and beads applied to a μColumn in the magnetic field of a μMACS magnetic separator. Beads were washed twice with pull-out wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). 4SU-RNA was eluted twice by addition of 100 mM DTT and RNA cleaned up using the RNeasy MinElute kit (Qiagen, 74204).
Libraries were prepared using the KAPA RNA HyperPrep kit (Roche) using 5 min 65°C fragmentation incubation to anneal primers but to prevent further fragmentation of the samples.

treatment_protocol: 2h of HS followed by either 10 min 1mM 4-thiouridine pulse or a treatment with 300nM Flavopiridol for 15, 20, 25, 30 or 40 min (including 10 min 1mM 4-thiouridine pulse).

value: 2h HS, followed by 40min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: ctrl_4SU.mRNA-seq_rep1

description:
mRNA-Seq
poly(A)+ mRNA


title: EtOH_GBS1eKO_4Cseq [GBS1eKO_EtOH_4C.rep1]

value: EtOH


title: EtOH_GBS1eKO_4Cseq [GBS1eKO_EtOH_4C.rep2]


title: Dex1hr_GBS1eKO_4Cseq [GBS1eKO_Dex_4C.rep2]


title: Dex1hr_GBS1eKO_4Cseq [GBS1eKO_Dex_4C.rep1]


title: ctrl_4SU.mRNA-seq_rep3

description:
mRNA-Seq
poly(A)+ mRNA


title: EtOH_GBS1eKO_4Cseq [GBS1eKO_EtOH_4C.rep3]


title: Dex1hr_A12_4Cseq [A12_Dex_4C.rep3]

value: A1-2 breast cancer cells


title: Dex1hr_A12_4Cseq [A12_Dex_4C.rep2]


title: CWRR1 Untreated #1

value: CWR-R1 cell line

value: castration-resistant


title: HS_FP.TTchem-seq_25min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 2h HS, followed by 25min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: CWRR1-Input, ChIP-Seq

source_name: prostate carcinoma cell line

value: prostate carcinoma epithelial cell line derived from a xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft


title: DVG-CWRR1, SOX2-ChIP-Seq


title: EtOH_GBS2eKO_4Cseq [GBS2eKO_EtOH_4C.rep2]


title: HS_4SU.mRNA-seq_rep3

description:
mRNA-Seq
poly(A)+ mRNA


title: EtOH_GBS2eKO_4Cseq [GBS2eKO_EtOH_4C.rep1]


title: Dex1hr_A12_4Cseq [A12_Dex_4C.rep1]

value: 1hr Dex


title: HS_FP.TTchem-seq_20min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 2h HS, followed by 20min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: HS_FP.TTchem-seq_15min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 2h HS, followed by 15min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: EtOH_A12_4Cseq [A12_EtOH_4C.rep3]


title: HS_FP.TTchem-seq_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 2h HS including 10 min 1mM 4-thiouridine pulse


title: HS_FP.TTchem-seq_40min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA


title: EtOH_A12_4Cseq [A12_EtOH_4C.rep2]


title: HS_FP.TTchem-seq_30min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA

value: 2h HS, followed by 30min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: HS_FP.TTchem-seq_25min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA


title: HS_FP.TTchem-seq_20min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA


title: HS_FP.TTchem-seq_15min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA


title: HS_FP.TTchem-seq_30min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA


title: ctrl_FP.TTchem-seq_40min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 40min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: C29-ALDU1MINUSUL

city: State College

first: Aswathy

last: Sebastian

address: University Park

institute: PennState

data_processing:
125 base pair paired-end reads were sequenced. Quality reports were generated with FastQC.
Adapter sequence and low quality bases were trimmed using trimmomatic. Reads with length <35 bp after trimming were discarded.
Trimmed data was mapped to the Gallus_gallus.GRCg6a assembly using hisat2 program. Coverage files were generated and alignments were visualized in IGV.
Transcript level abundances were genereated by the pseudoalignment of the dataset to the chicken transcriptome using Kallisto.Transcript level read counts and TPMs were generated.
Transcript level raw read counts were normalized and differentially expressed transcripts were identified using DESeq2 package in R.
Genome_build: Gallus_gallus.GRCg6a
Supplementary_files_format_and_content: Transcript-level raw counts and TPMs
Supplementary_files_format_and_content: Deseq2 normalized counts and differential expression results

organism: Gallus gallus

source_name: COVCAR cells designated as C29 SORTED FOR ALDH- CELLS Replicate 2

growth_protocol: COVCAR cells were cultured on gelatinized 75cm cell culture flasks at 37ºC under 5% CO2 atmosphere.  When the COVCAR cells reached 100% confluence, the cells were dissociated from the flask using 0.05% trypsin (Invitrogen) and used for sorting ALDH+ and ALDH- cells

extract_protocol:
Total RNA was extracted using the RNeasy  kit (Qiagen) using the manufacturer’s protocol.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Approximately 5 million COVCAR cells from each of 3 primary ovarian cancer lines designated as C27, C29, and C30 were sorted using FACS for the presence of ALDH activity. Cells sorted for ALDH+ or ALDH- were seperated and total RNA was extracted.

value: Ovarian cancer cells derived fom ascites of chickens with ovarian cancer

tag: tumor type

value: Ovarian adenocarcinoma

value: ALDH- cells


title: C29-ALDU1PLUSUL

source_name: COVCAR cells designated as C29 SORTED FOR ALDH+ CELLS Replicate 2

value: ALDH+ cells


title: C30-ALDU1MINUSUL

source_name: COVCAR cells designated as C30 SORTED FOR ALDH- CELLS Replicate 3


title: C30-ALDU1PLUSUL

source_name: COVCAR cells designated as C30 SORTED FOR ALDH+ CELLS Replicate 3


title: ctrl_FP.TTchem-seq_30min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 30min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: ctrl_FP.TTchem-seq_25min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 25min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: ctrl_FP.TTchem-seq_20min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 20min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: ctrl_FP.TTchem-seq_15min_FP_rep2

description:
FP/TTchem-seq
Nascent RNA

value: 15min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: ctrl_FP.TTchem-seq_rep2

description:
FP/TTchem-seq
Nascent RNA

value: Control, 10 min 1mM 4-thiouridine pulse


title: EtOH_A12_4Cseq [A12_EtOH_4C.rep1]

value: A1-2 breast cancer cells

value: EtOH

value: DDIT4 TSS


title: ctrl_FP.TTchem-seq_40min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA


title: C27-ALDU1PLUSUL

source_name: COVCAR cells designated as C27 SORTED FOR ALDH+ CELLS Replicate 1


title: ctrl_FP.TTchem-seq_25min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA


title: C27-ALDU1MINUSUL

source_name: COVCAR cells designated as C27 SORTED FOR ALDH- CELLS Replicate 1


title: HS_FP.TTchem-seq_rep1

description:
FP/TTchem-seq
Nascent RNA


title: ctrl_FP.TTchem-seq_15min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA


title: ctrl_FP.TTchem-seq_rep1

description:
FP/TTchem-seq
Nascent RNA


title: HS_DRB.TTchem-seq_40min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

extract_protocol:
RNA was extracted by TRIzol (Thermo Fisher Scientific, 15596026) accordingly to the manufacturer’s instructions. As a control for equal sample preparation, we spiked-in S. cerevisiae (strain BY4741, MATa, his3D1, leu2D0, met15D0, ura3D0) 4-thiouracil (4TU)-labelled RNA. S. cerevisiae were grown in YPD medium overnight, diluted to an OD600 of 0.1, and grown to mid-log phase (OD600 of 0.8) and labelled with 5 mM 4TU (Sigma-Aldrich, 440736) for 6 min. Total RNA was extracted using the PureLink RNA Mini kit (Thermo Fisher Scientific, 12183020) following the enzymatic protocol. For purification of 4SU labelled RNA, 100 μg mammalian 4SU labelled RNA was spiked-in 1/100 of 4TU-labelled S. cerevisiae RNA. The 101 μg RNA was fragmented by addition of 20 μL 1 M NaOH to fragment RNA and left on ice for 20 min. Fragmentation was stopped by addition of 80 μL 1 M Tris pH 6.8 and cleaned up twice with Micro Bio-Spin™ P-30 Gel Columns (BioRad, 7326223) according to the manufacturer’s instructions. Biotinylation of 4SU-residues were carried out in a total volume of 250 μl, containing 10 mM Tris-HCl [pH 7.4], 1 mM EDTA and 5mg MTSEA biotin-XX linker (Biotium, BT90066) for 30 min at room temperature in the dark. RNA was then purified by phenol:chloroform extraction, denatured by 10 min incubation at 65°C and added to 200 μL μMACS Streptavidin MicroBeads (Miltenyi, 130-074-101). RNA was incubated with beads for 15 min at room temperature and beads applied to a μColumn in the magnetic field of a μMACS magnetic separator. Beads were washed twice with pull-out wash buffer (100 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween20). 4SU-RNA was eluted twice by addition of 100 mM DTT and RNA cleaned up using the RNeasy MinElute kit (Qiagen, 74204).
Libraries were prepared using the KAPA RNA HyperPrep kit (Roche) using 5 min 65°C fragmentation incubation to anneal primers but to prevent further fragmentation of the samples.

treatment_protocol: 1.5 hrs pre-treatment with 100uM DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole), followed by 2h of DRB treatment in Heat Shock conditions and either 10 min 1mM 4-thiouridine pulse or release from DRB inhibition for 10, 20, 30 or 40 min (including 10 min 1mM 4-thiouridine pulse).

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, including HS for 2h, release from DRB inhibition for 40 min (including 10 min 1mM 4-thiouridine pulse)


title: HS_DRB.TTchem-seq_30min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, including HS for 2h, release from DRB inhibition for 30 min (including 10 min 1mM 4-thiouridine pulse)


title: Treatment 14 days replicate 3

type: RNA

city: Madrid

first: Pedro

last: Botias

address: Jose Antonio Nováis 12

country: Spain

institute: Universidad Complutense de Madrid

hyb_protocol: RNA was hybridized to GeneChip C. elegans Gene 1.0 ST Arrays following manufacturers instructions (Affymetrix, Santa Clara, CA) in a GeneChip Hybridization Oven 645 at 45ºC with rotation (60 rpm). The hybridized samples were washed and stained using an Affymetrix fluidics station 450.

scan_protocol: After washing and staining, the probe array was scanned using Affymetrix Genechip scanner GCS3000 7G system and the software Genechip Command Console Software (AGCC)

data_processing: The intensity cell files were then imported, normalized for background correction (used RMA for normalization) with Transcriptome Analysis Console (TAC)

label: Biotin

organism: Caenorhabditis elegans

source_name: nematodes

label_protocol: RNA was labeled using the GeneChip WT PLUS Reagent kit (Affymetrix, High Wycombe, UK).

growth_protocol: The C. elegans wild type strain N2 was provided by the Caenorhabditis Genetic Center (University of Minnesota, St. Paul, MN, USA). The strain was maintained on nematode growth medium (NGM) plates seeded with Escherichia coli strain OP50 at 20 ºC (Brenner, 1974). Chunks of agar starved plates with worms from stocks of dauer larvae were transferred to fresh NGM plates seeded with OP50. After 3 days many gravid hermaphrodites, as well as L1 and L2 stage juveniles, were found on the plates. Age-synchronization of worms was achieved by washing them off the plates and filtered with K-medium. A 10 µm nylon net filter (Merck Millipore Ltd. Cork, IRL), which retains all but L1-L2 juvenile stage nematodes, was used.

extract_protocol: RNA was extracted from each sample replicate using RNeasy Mini Kit (Qiagen, Iberia, S.L) following the manufacturer's protocols.

treatment_protocol: L1-L2 worms were allowed to develop 2 days at 20 ºC in liquid culture to obtain the L4 stage and young adults. Then, the density of the nematodes was adjusted to 15000-20000 worms per mL. To each test well of the 12-well microtiter plate, 0.3 g of soil containing HMs (2700 mg kg-1, 4200 mg kg-1, and 310 mg kg-1 of Pb, Zn, and Cd, respectively), with or without nZVI, were added and  moistened with 0.2 mL of K-medium, respectively. In addition, 0.25 mL of E.coli OP50 (2.5 x 1010 cell mL-1, in K-medium) and 0.5 mL (7500-10000 worms) from the nematode suspension were added, mixed with the soil, and  incubated 48 h at 20 ºC in the dark. The recovery of the nematodes was carried out by using the suspension of colloidal silica (Ludox). The content of the wells was transferred into 50 mL tubes and centrifuged (5 min, 800 x g). The supernatant with nematodes was filtered (10 µm nylon net filter) to remove L1 larvae, rinsed back into the tubes and newly centrifuged (15 min, 800 x g). The collected nematodes therefore consist of young adult worms. The pellet was suspended in buffer lysis and stored at -20 ºC until RNA extraction.

tag: strain

value: wild type strain N2

value: nZVI-treated soil

tag: timepoint

value: 14 days


title: Treatment 4 months replicate 1

value: 4 months


title: Treatment 4 months replicate 2


title: Treatment 4 months replicate 3


title: Treatment 14 days replicate 1


title: Treatment 14 days replicate 2


title: HS_DRB.TTchem-seq_20min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, including HS for 2h, release from DRB inhibition for 20 min (including 10 min 1mM 4-thiouridine pulse)


title: Treatment replicate 1

value: 0 days


title: HS_DRB.TTchem-seq_10min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, including HS for 2h, release from DRB inhibition for 10 min (including 10 min 1mM 4-thiouridine pulse)


title: Treatment replicate 3


title: HS_DRB.TTchem-seq_0min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs including HS for 2h and 10 min 1mM 4-thiouridine pulse


title: Control 4 months replicate 2

value: control


title: Control 4 months replicate 1


title: Control 14 days replicate 3


title: ctrl_FP.TTchem-seq_30min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA

value: 30min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: ctrl_FP.TTchem-seq_20min_FP_rep1

description:
FP/TTchem-seq
Nascent RNA

value: 20min of treatment with 300nM Flavopiridol (including 10 min 1mM 4-thiouridine pulse)


title: HS_DRB.TTchem-seq_40min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA


title: Treatment replicate 2


title: Control 14 days replicate 2


title: HS_DRB.TTchem-seq_30min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA


title: HLKLYBBXX_15803806_S63_L007

city: Bethesda

first: David

last: McGaughey

state: MD

address: 10 Center Dr

department: NEI

institute: NIH

data_processing:
Macaca_mulatta.Mmul_8.0.1.90.gtf.gz used as reference for Salmon index build
Quantified with Salmon 0.13
Imported into R with tximport (lengthScaledTPM)
HCOP annotation information used to align gene names with ensembl ID
TPM values scaled for library size with edgeR calcNormFactors
Genome_build: Macaca_mulatta.Mmul_8.0.1.90.gtf.gz
Supplementary_files_format_and_content: TSV

organism: Macaca mulatta

extract_protocol: Two-millimeter punch biopsies were removed from macular/foveal, perimacular, temporal, and nasal regions of the retina.  Following the biopsy, the tissues were separated into layers of neural retina and RPE/choroid using microdissection techniques. otal RNA was isolated from fresh tissues using the PicoPure™ RNA Isolation Kit (Applied Biosystems, Waltham, Massachusetts, USA) following mortar and pestle tissue homogenization.  Extracted RNA samples were checked for quality using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and only samples with RIN value > 6.5 were used (Range 1 – 10).

treatment_protocol: Postmorterm rhesus

tag: tissue

value: RPE-Choroid

tag: location

value: Peripheral

tag: eye (od/os)

tag: macaque number


title: HS_DRB.TTchem-seq_0min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA

tag: cell line

tag: treatment


title: HS_DRB.TTchem-seq_10min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA


title: HLKLYBBXX_15803806_S63_L008


title: HLKLYBBXX_15803800_S57_L007

value: Retina


title: Control replicate 3


title: ctrl_DRB.TTchem-seq_40min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, release from DRB inhibition for 40 min (including 10 min 1mM 4-thiouridine pulse)


title: Control 14 days replicate 1


title: Control replicate 1


title: Control 4 months replicate 3


title: HLKLYBBXX_15803801_S58_L007


title: HLKLYBBXX_15803804_S61_L007


title: HLKLYBBXX_15803801_S58_L008


title: InPut 4w

city: Chengdu

first: Wenjing

last: Zhang

state: Sichuan

address: Keyuan Road 4,Gaopeng Street

department: Regenerative Medicine Research Center

institute: West China Hospital,Sichuan University

data_processing:
Reads filtering under criteria removing reads with 20% of the base quality lower than 13.
ChIP-seq reads were aligned to the GCF_000772875.2 genome assembly using Bowtie aligner version 2.0 with default parameters.
Data were filtered using the following specifications
MACS2 peak finding algorithm (version 2.1.0) was used to identify regions of ChIP-seq enrichment over background (pvalue < 0.01).
Genome_build: GCF_000772875.2
Supplementary_files_format_and_content: peaks were called using macs2.

source_name: myocardial infarction_4 wks

growth_protocol: All Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care accredited facility and were treated in strict accordance with good animal practice under supervision of veterinarians.

extract_protocol: Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody.

treatment_protocol: Hearts were removed, flash frozen and grinded in nitrogen. And fixed in 1% formaldehyde.Homogenate tissue in a Dounce homogenizer.

tag: Sex

value: Male

tag: model

value: Myocardial ischemic infarction

value: heart

tag: time point

value: 4 weeks

tag: chip-antibody


title: ctrl_DRB.TTchem-seq_30min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, release from DRB inhibition for 30 min (including 10 min 1mM 4-thiouridine pulse)


title: InPut 6h

source_name: myocardial infarction_6hrs


title: HLKLYBBXX_15803804_S61_L008


title: ctrl_DRB.TTchem-seq_20min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, release from DRB inhibition for 20 min (including 10 min 1mM 4-thiouridine pulse)


title: ctrl_DRB.TTchem-seq_10min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, release from DRB inhibition for 10 min (including 10 min 1mM 4-thiouridine pulse)


title: Control replicate 2

value: wild type strain N2

value: 0 days


title: HS_DRB.TTchem-seq_20min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs, including HS for 2h, release from DRB inhibition for 20 min (including 10 min 1mM 4-thiouridine pulse)


title: MI 4 w

value: Anti-HIF-1 alpha (abcam, ab1, Lot GR194781-3)


title: MI 6 h


title: ctrl_DRB.TTchem-seq_0min_release_rep2

description:
DRB/TTchem-seq
Nascent RNA

value: 100uM DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) for 3.5 hrs (including 10 min 1mM 4-thiouridine pulse)


title: HLKLYBBXX_15803800_S57_L008


title: HLKLYBBXX_15803802_S59_L008


title: HLKLYBBXX_15803803_S60_L007


title: HLKLYBBXX_15803805_S62_L008


title: HLKLYBBXX_15803805_S62_L007


title: HLKLYBBXX_15803802_S59_L007


title: HLKLYBBXX_15803807_S64_L008


title: ctrl_DRB.TTchem-seq_30min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA


title: HLKLYBBXX_15803807_S64_L007


title: ctrl_DRB.TTchem-seq_40min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA


title: HLKMNBBXX_15771291_S13_L002

value: Nasal


title: ctrl_DRB.TTchem-seq_20min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA


title: HLKJ7BBXX_15771291_S1_L001

value: RPE-Choroid


title: HLKMNBBXX_15771291_S13_L003


title: HLKMNBBXX_15771291_S13_L004


title: HLKJ7BBXX_15771311_S21_L002


title: HLKJ7BBXX_15771291_S1_L002


title: HLKLYBBXX_15803803_S60_L008

value: Peripheral


title: ctrl_DRB.TTchem-seq_0min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA


title: ctrl_DRB.TTchem-seq_10min_release_rep1

description:
DRB/TTchem-seq
Nascent RNA


title: HLKMNBBXX_15771311_S33_L002

value: Retina


title: HLKMNBBXX_15771311_S33_L003


title: HLKMNBBXX_15771311_S33_L004


title: MRC5VA_HS60min_tc_TTchem-seq_rep2

description:
TTchem-seq
Nascent RNA

treatment_protocol: Nascent RNA labelling by addition of 1 mM 4-thiourdine to cell media for 15 min prior to harvest.

value: 60min Heat Shock, 15 min 4-thiouridine pulse


title: HLKJ7BBXX_15771311_S21_L001


title: MRC5VA_HS180min_tc_TTchem-seq_rep2

description:
TTchem-seq
Nascent RNA

value: 180min Heat Shock, 15 min 4-thiouridine pulse


title: HLKJ7BBXX_15771308_S18_L002


title: HLKMNBBXX_15771308_S30_L002


title: HLKMNBBXX_15771308_S30_L003


title: HLKJ7BBXX_15771308_S18_L001


title: HLKMNBBXX_15771308_S30_L004


title: HLKMNBBXX_15771312_S34_L002


title: set2 Ioc4 Flag-IP Replicate 3

city: Martinsried-Planegg

first: Michaela

middle: M

last: Smolle

address: Grosshaderner Street 9

department: Physiological Chemistry

institute: University of Munich

description: Biological Replicate 3 of 3. ChIP enrichment of FLAG-IP over Input in the YMS087 IOC4-3xFLAG set2 strain.

hyb_protocol: The hybridization mixture (Oligo CGH/Chip-on-chip hybridization kit, Agilent) was prepared according to manufacturer’s instructions with the addition of 20 ug of T7 blocking oligo (ttt ttt ttt ttt tt cac gcg tct ccc), and hybridized overnight at 65°C. Microarrays were washed using the Oligo aCCH/Chip-on-chip Wash buffers (Agilent) according to  manufacturer’s instructions.

scan_protocol: Scanned on Agilent Technologies Scanner G2505C US45102919 Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).

data_processing: Data processed in R (version 2.13.0) using Bioconductor: MEDIAN normalization.

organism: Saccharomyces cerevisiae

source_name: IP DNA FLAG antibody

label_protocol: Briefly, up to 50 ng of IP or input DNA was treated with 2.5 U CIP enzyme (NEB) for 1 hour at 37°C, followed by phenol:chloroform extraction and ethanol precipitation. The CIP-treated template was incubated with 20 U TdT (NEB) for 20 minutes at 37C for T-tailing the ends, and the product isolated with the MinElute Reaction Cleanup Kit (Qiagen). An anchored T7 promoter-(dA)18 oligo was added by annealing and filling with 5 U Exo- Klenow (NEB) for 4 hours at 37°C, followed by phenol:chloroform extraction and ethanol precipitation. In vitro transcription was performed at 37°C overnight using the Ampliscribe T7 transcription kit (Epicentre Biotechnologies). RNAs purified using the RNeasy Mini Kit (Qiagen) and quantified on a Nanodrop 2000 Spectrophotometer (Thermo Scientific). For the second round amplification, 100 ng of RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen) and purified with the MinElute Reaction Cleanup Kit (Qiagen). The T7 promoter primer was added to the product through a Klenow filling reaction as described above, followed by in vitro transcription with amino allyl-UTP (Ambion) instead of UTP. Final reaction cleanup was performed with the RNeasy Mini Kit (Qiagen). For the labeling reactions, 8 ug of amino allyl-incorporated RNA in a 5 uL volume of 0.1 M Carbonate Buffer, pH 8.7, was mixed with 5 uL (0.01 nmol) Cy3 or Cy5 dye (GE Healthcare) in DMSO (Sigma) and incubated at room temperature for 2 hours. Reactions were quenched with 5 uL 4 M hydroxylamine at room temperature for 15 minutes, purified by RNeasy MinElute Cleanup Kit (Qiagen), and the dye incorporation measured using the Nanodrop 2000 (Thermo Scientific). 4ug of input and IP were combined and fragmented (Fragmentation Reagent Kit, Ambion) according to manufacturer’s instructions.

growth_protocol: These strains were grown in YPD to an OD of 1.0. Cells were cross-linked and processed for the ChIP assay. The cell lysates were used to immunoprecipitate 3xFLAG tagged wildtype and mutant Ioc4 (2 ul αFLAG per IP).

extract_protocol: Lysates were prepared by bead beating in FA lysis buffer (50mM HEPES pH7.5, 1mM EDTA, 1% Triton X-100, 0.1%sodium deoxycholate and 150mM NaCl). The ChIP lysates were used to immunoprecipitate FLAG-tagged Ioc4. The immune complexes were pulled down with 50 ul of Protein G Dynabeads (Invitrogen) per IP and washed with FA lysis buffer, FA lysis buffer with 1M NaCl, FA lysis buffer with 0.5M NaCl, TEL buffer (0.25M LiCl, 1%NP-40, 1% sodium deoxycholate, 1mM EDTA, 10mM TrisHCL, pH8.1), and twice with TE. The Immune complexes were eluted with Elution buffer (1%SDS in TE with 250mM NaCl). The eluates were RNase A treated and Proteinase K was added to remove the bound proteins. Crosslinkss were reversed by incubating at 65°C overnight. Samples werte phenol:chloroform treated and ethanol precipitated. Input samples were treated the same way from the RNase A step.

tag: sample type

value: ChIP IP

source_name: Input DNA

value: ChIP Input


title: MRC5VA_HS180min_tc_TTchem-seq_rep1

description:
TTchem-seq
Nascent RNA


title: set2 Ioc4 Flag-IP Replicate 2

description: Biological Replicate 2 of 3. ChIP enrichment of FLAG-IP over Input in the YMS087 IOC4-3xFLAG set2 strain.


title: MRC5VA_ctrl_tc_TTchem-seq_rep2

description:
TTchem-seq
Nascent RNA

value: Control, 15 min 4-thiouridine pulse


title: HLKJ7BBXX_15771312_S22_L002


title: set2 Ioc4 Flag-IP Replicate 1

description: Biological Replicate 1 of 3. ChIP enrichment of FLAG-IP over Input in the YMS087 IOC4-3xFLAG set2 strain.


title: HLKMNBBXX_15771312_S34_L004


title: Ioc4-dINS Flag-IP Replicate 3

description: Biological Replicate 3 of 3. ChIP enrichment of FLAG-IP over Input in the YMS255 IOC4dINS-3xFLAG strain.


title: Ioc4-dINS Flag-IP Replicate 2

description: Biological Replicate 2 of 3. ChIP enrichment of FLAG-IP over Input in the YMS255 IOC4dINS-3xFLAG strain.


title: Ioc4-2KE Flag-IP Replicate 3

description: Biological Replicate 3 of 3. ChIP enrichment of FLAG-IP over Input in the YMS265 IOC42KE-3xFLAG strain.


title: Ioc4-dINS Flag-IP Replicate 1

description: Biological Replicate 1 of 3. ChIP enrichment of FLAG-IP over Input in the YMS255 IOC4dINS-3xFLAG strain.


title: HLKJ7BBXX_15771292_S2_L002


title: MRC5VA_ctrl_tc_TTchem-seq_rep1

description:
TTchem-seq
Nascent RNA


title: HLKMNBBXX_15771292_S14_L004


title: MRC5VA_HS60min_tc_TTchem-seq_rep1

description:
TTchem-seq
Nascent RNA


title: MRC5VA_HS30min_tc_TTchem-seq_rep1

description:
TTchem-seq
Nascent RNA

value: 30min Heat Shock, 15 min 4-thiouridine pulse


title: MRC5VA_HS30min_tc_TTchem-seq_rep2

description:
TTchem-seq
Nascent RNA


title: HLKMNBBXX_15771292_S14_L002


title: HLKMNBBXX_15771292_S14_L003


title: Ioc4-2KE Flag-IP Replicate 1

description: Biological Replicate 1 of 3. ChIP enrichment of FLAG-IP over Input in the YMS265 IOC42KE-3xFLAG strain.


title: HLKJ7BBXX_15771304_S14_L001


title: HLKJ7BBXX_15771304_S14_L002


title: MRC5VA_HS120min_TTchem-seq_rep2

description:
TTchem-seq
Nascent RNA

value: 120min Heat Shock, 15 min 4-thiouridine pulse


title: MRC5VA_ctrl_TTchem-seq_rep2

description:
TTchem-seq
Nascent RNA


title: Ioc4-dPWWP Flag-IP Replicate 2

description: Biological Replicate 2 of 3. ChIP enrichment of FLAG-IP over Input in the YMS264 IOC4dPWWP-3xFLAG strain.


title: HLKJ7BBXX_15771312_S22_L001


title: Ioc4-2KE Flag-IP Replicate 2

description: Biological Replicate 2 of 3. ChIP enrichment of FLAG-IP over Input in the YMS265 IOC42KE-3xFLAG strain.