title: Dt47.day6.AZA.0.5

data_processing:
Library strategy: Bisulfite-treated Amplicon-seq
Fastq files were aligned to two custom-made human genomes covering the b-globin locus only: (1) a genome in which all the cytosines (C) are converted to uracil (U), read as thymine (T) by the Illumina sequencer; (2) a genome which is the wild-type human genome of the b-globin locus.
DNA sequences are expected to partially align to the custom-made genomes, depending on the extent of bisulfite conversion. Because perfect alignment will not be expected due to differing methylation levels across the CpGs, we scanned for partially aligned sequences and subject them to a second step of counting.
For this second step, a computer program was written to locally align each sequence to the entire amplicon again, but this time counted the number of converted cytosines at each of 8 CpG loci, which is seen as a mismatched base T compared to the wildtype genome in the local alignment.
Methylation frequencies were computed at each CpG locus as the number of aligned sequences containing the converted cytosine at the locus, normalized by the total number of aligned sequences covering the locus (the sum of converted cytosines and unconverted cytosines)
Sequences were first trimmed using Trimmomatic (v0.36) with parameters: 2:15:4:4:true LEADING:20 TRAILING:20  SLIDINGWINDOW:4:15 MINLEN:25. Alignment was performed using Bowtie2 (version 2.2.9) with parameters -p 2 –dovetail –phred33 using the custom genome.
Genome_build: custom genome hg19
Supplementary_files_format_and_content: The processed file list the DNA methylation status of each of 8 CpG loci in the b-globin locus. For each sequence, it shows three columns: column 1 is the CpG genomic position (along the b-globin locus), column 2 is the wildtype sequence (it is always C), column 3 is the methylation status (T if C is unmethylated; C if C is methylated).

treatment_protocol: Cells were resuspended in medium containing 0.15uM dTAG-47. Every other day, cells were centrifuged at 500g for 5min at 4C and resuspended in fresh media containing the drug. HUDEP-2 cells were cultured in differentiating media containing DMSO or 0.15dTAG-47 in addition to 0M, 0.1M, or 0.3M Azacytidine. Cells were spun down and re-suspended in fresh media containing the drugs owing to the short half-life of AZA in cell culture. 0.5-1x106 cells were collected, flash-frozen in LN2, and stored at -80oC

tag: aza concentration


title: plate2_F11_L002_AK16886-GH10_RNAseq

status: Public on Jul 19 2022

type: SRA

city: Cambridge

first: Felix

last: Krueger

address: Granta Park

department: Bioinformatics

country: United Kingdom

institute: Altos Labs

library_source: transcriptomic

data_processing:
Libraries were sequenced on the Illumina Nextseq500 platform using the default RTA analysis software.
RNA-seq libraries were aligned to the GRCm38 mouse genome build using HISAT2 (v2.1.0) using options --dta --no-softclipping --no-mixed --no-discordant, yielding a mean of 611,000 aligned reads per cell.
Subsequently, gene expression counts were quantified from the mapped reads using featureCounts with the Ensembl gene annotation (version 87). Only protein-coding genes matching canonical chromosomes were considered. The read counts were log-transformed and size-factor adjusted.
Assembly: GRCm38
Supplementary files format and content: A table of genes (rows) x cells (columns). The first column is the ensembl gene ID

molecule: total RNA

organism: Mus musculus

source_name: Early embryo

extract_protocol: Single-cells were sorted  using flow cytometry (E6.5, E7.5) into 96 well PCR plates containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL (Sigma)). Samples were incubated for 15 minutes at 37°C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at -80°C prior to processing. Poly-A RNA was captured on oligo-dT conjugated to magnetic beads and amplified cDNA was prepared according to the G&T-seq and Smartseq2 protocols. The lysate containing gDNA was purified on AMPureXP beads before bisulfite-seq libraries were prepared according to the scBS-seq protocol.

tag: strain

tag: tissue

value: E7.5 embryo


title: E8.5_plate2_H09_RNAseq

value: E8.5 embryo


title: plate2_G04_L002_AK7737-AK903_RNAseq


title: plate2_F12_L002_AK3914-GH10_RNAseq


title: Dt47.day6.AZA.0.3

city: Boston

first: Stuart

middle: H

last: Orkin

state: MA

address: 1 Blackfan Circle KFRB 07007H

department: Hematology/Oncology

country: USA

institute: Dana-Farber Cancer Institute/Boston Children's hospital

library_source: genomic

molecule: genomic DNA

organism: Homo sapiens

source_name: HUDEP2 cells

growth_protocol: HUDEP-2 lines (human umbilical cord blood-derived erythroid progenitor, male) were provided by Dr. Yukio Nakamura. HUDEP-2 cells were cultured as described previously (Vinjamur and Bauer 2018). Briefly, cells were maintained at a density of <0.5x106 in expansion medium: StemSpan™ SFEM (STEMCELL Technologies, 09650) with SCF (50 ng/ml), EPO (3 IU/ml), Dexamethasone (10−6 M), Doxycycline (1 μg/ml), and passaged every other day. Erythroid differentiation was carried out by replacing the medium to EDM2 (Erythroid Differentiation Media: IMDM (Corning, 15–016-CV) supplemented with 330 μg/mL HoloHuman Transferrin, 10 μg/mL Recombinant Human Insulin, 2 IU/mL Heparin, 5% Inactivated Plasma, 3 IU/mL Erythropoietin, 2 mM L-Glutamine, 100 ng/mL SCF, 1 μg/mL doxycycline).

extract_protocol:
On the day of the experiment, DNA was extracted from frozen pellets with an Allprep DNA/RNA Mini Kit (Qiagen). Bisulfite conversion was performed on 1 μg DNA with an Epitect kit (Qiagen), and a 457bp region of the HBG1/2 promoter region was amplified by nested PCR as described previously (Mehta et al. 2015).
PCR amplicons were sent for Illumina Amplicon next-generation sequencing (Genewiz), yielding 2x250 bp reads.

tag: cell line

tag: day

tag: treatment


title: Dt47.day6.AZA.0


title: E8.5_plate2_H07_RNAseq


title: E8.5_plate2_H08_RNAseq


title: hsc_pIpC_2

city: Heidelberg

first: Sina

last: Stäble

address: Im Neuenheimer Feld 280

department: Translational Cancer Epigenomics

country: Germany

institute: German Cancer Research Center (DKFZ)

description: bisulfite converted genomic DNA

data_processing:
Sequence alignment was performed using an updated version of the workflow published in (Wang et al., Nat. Prot., 2013). The workflow was implemented as a Roddy Workflow  (https://github.com/DKFZ-ODCF/AlignmentAndQCWorkflows). Alignment was performed against the mm10 reference genome extended with the PhiX and lambda phage sequences. Briefly, before alignment, trimmomatic was used for adapter trimming and reads were in silico converted (C>T for the first read in the pair, G>A for the second). Alignments of the individual libraries were performed against the concatenated C>T and G>A in silico converted reference genomes using bwa mem with default parameters. After alignment, reads were converted back to their original state.
PCR duplicate removal was performed per library using Picard MarkDuplicates before merging reads from all libraries.
At each cytosine position, reads that maintain the cytosine status were considered methylated, and the reads that have cytosine converted to thymine were considered unmethylated.
Only reads with a mapping quality >= 25 and bases with Phred-scaled quality score ≥25 were considered for methylation calling.
At least the first 9 bp of the second read and last 9 bp before the adapter in the first read were excluded from methylation calling. The in silico conversion and methylation calling steps were performed using bistro (https://github.com/stephenkraemer/bistro).
Assembly: mm10
Supplementary files format and content: QC-filtered methylation calls for all autosomal CpGs in BED6+3 format, with the following columns: chrom, start, end, motif, score, strand, beta_value, n_meth, n_total. The two cytosines in a CpG motif were merged, and the CpG position is given on the plus strand. The score column holds no information (always '.').

source_name: LT-HSC

extract_protocol:
To isolate purified LT-HSCs from the murine bone marrow, density gradient centrifugation was performed to enrich low-density mononuclear cell (LDMNCs), followed by lineage depletion of mature hematopoietic cell lineages using rat anti-mouse biotin-conjugated lineage antibodies and Biotin Binder Dynabeads (Life Technologies). LT-HSCs were then isolated from lineage-negative bone marrow cells by fluorescence activated cell sorting. DNA was isolated from snap-frozen cell pellets (15.000 - 20.000 sorted cells) using the QIAamp DNA Micro Kit (Qiagen) according to the manufacturer’s instructions.
Tagmentation-based whole-genome bisulfite sequencing (TWGBS) libraries were prepared as described previously (Wang et al., Nat. Prot., 2013). We used 10-30 ng genomic DNA as input.

treatment_protocol: To mimic a repetitive sterile inflammatory response in vivo, wild-type C57BL/6J mice were subjected to a three round pI:pC dose escalation regimen. Mice were serially injected intraperitoneally (i.p.) with 5 mg/kg high molecular weight polyinosinic:polycytidylic acid (pI:pC, InvivoGen), which had been reconstituted in sterile physiologic saline precisely as described in the manufacturer’s instructions. Control mice were injected with the same volume of PBS (Sigma Aldrich). One round of pI:pC treatment corresponds to 8 individual injections spread over the course of 4 weeks, followed by a 4 week recovery period prior to the next round of treatment.

tag: cell type

tag: age

value: 32-36 weeks

value: pIpC


title: plate2_F10_L002_AK7505-GH10_RNAseq


title: hsc_pIpC_1

description: bisulfite converted genomic DNA


title: plate2_F09_L002_AK3910-GH10_RNAseq


title: E8.5_plate2_H05_RNAseq


title: Dt47.day6.AZA.0.1


title: hsc_pbs_3

description: bisulfite converted genomic DNA

value: PBS


title: E8.5_plate2_H06_RNAseq


title: Dt47.day4.AZA.0


title: plate2_F08_L002_AK2870-GH10_RNAseq


title: hsc_pbs_2

description: bisulfite converted genomic DNA


title: Dt47.day4.AZA.0.5


title: E8.5_plate2_H04_RNAseq


title: plate2_F07_L002_AK9176-GH10_RNAseq


title: Dt47.day4.AZA.0.3


title: plate2_F05_L002_AK9173-GH10_RNAseq


title: hsc_pbs_1

description: bisulfite converted genomic DNA


title: Liver GH3:328G

city: Matsudo

first: Akihito

last: Yausoka

state: Chiba

address: Iwase 550

department: Human nutrition

country: Japan

institute: Seitoku University

data_processing:
mRNA-seq:
The Fastq data were checked by Fastqc quality check program and trimmed using the fastx-clipper and cutadapt program to remove adaptor sequence (>1%) and low-quality reads (quality < 30, length < 20 base). Cleaned reads were mapped against Mus musculus reference genome GRCm 38 (mm10, https://www.ncbi.nlm.nih.gov/assembly/GCF_000001635.20/) using tophat program (tophat-program: –ribrary-type FR second Strand -g20 -N2). The rate of mapped reads to the obtained reads was 96.5 % on average. The resulting BAM data were used to obtain the reads on the exons of known protein coding genes (mm10) on the featureCounts program (https://sourceforge.net/projects/subread/files/subread-2.0.0/). The reads per kilobase of exon model per million mapped reads (RPKM) for each tissue were filtered (RPKM < 0.3). The rate of filtered reads to the obtained reads was 56.8 % on average. Differentially expressed genes (DEGs) were predicted using the DESeq2 package on R statistical software (p < 0.01) (https://bioconductor.org/packages/release/bioc/html/DESeq2.html). Cluster dendrograms were drawn by hclust package (https://www.rdocumentation.org/packages/stats/versions/3.6.2/topics/hclust).
16S DNA-seq:
The processing of sequencing data, including quality filtering, chimera check, operational taxonomic unit (OTU) definition, and taxonomy assignment was carried out using the Quantitative Insights Into Microbial Ecology (QIIME software package version 1.8.0.), EA-Utils (ver. 1.1.2–537), the gold database (http://drive5.com/otupipe/gold.tz) and USEARCH (ver. 5.2.32, https://www.drive5.com/usearch/). Principal coordinate analysis, alpha diversity (Shannon index) analysis, and beta diversity analysis were conducted using vegan (Package vegan version 2.3-4) on R (2015, version 3.1.3) (Oksanen J et al., 2016).
Assembly: mm10; Quantitative Insights Into Microbial Ecology (QIIME software package version 1.8.0.)

source_name: Liver transcriptome

growth_protocol: Five-week-old (postnatal day 35, PD35) male C57BL/6N mice were procured from Charles River (Hino, Japan). The animals were shipped in cardboard cages (5 mice / 1 cage), housed in air conditioned rooms (22 ± 2 °C, and 50 ± 5% humidity), and subjected to a cyclic 12 h light (8 a.m.–8 p.m.) and dark (8 p.m.–8 a.m.) environment. The light intensity was 100 lx at the front of the cages and 40 lx behind the cages. Five mice were acclimatized in a cage (24 cm length × 17.2 cm width × 12.9 cm depth, 413 cm2 floor area) with free access to the chow diet (MF, Oriental yeast, Tokyo, Japan) and water until PD40 (Fig. 1).

extract_protocol:
mRNA-seq: Total RNA was isolated from amygdala, hypothalamus or liver using TRI Reagent (MRC molecular research center, OH, USA). Total RNA from the liver was further purified using SimplyRNA tissue kit on Maxwell RNA extraction system (Promega). RNA quality was accessed using RNA 6000 Nano Series II Kit on Agilent 2100 Bioanalyzer (Agilent141 Technologies, Santa Clara, CA, USA). RNA integrity number for each tissue RNA was higher than 8.5.
mRNA-seq: 200ng of Total RNA was constructed in a library using Smarter Stranded Total RNA Sample Prep Kit HI Mammalian (Clontech Laboratory, Inc.). The library was quantified using High Sensitivity DNA kit on Agilent 2100 Bioanalyzer and subjected to 50 bp single end sequencing analysis (55.7 million / sample on average) on a Hiseq system (Illumina).
16S DNA-seq: DNA was extracted from the cecal samples using the bead-beating method described by Odamaki et al (Odamaki T, Kato K, Sugahara H, Hashikura N, Takahashi S, Xiao JZ et al. (2016). Age-related changes in gut microbiota composition from newborn to centenarian: a cross-sectional study. BMC Microbiol. 16, 90.).
16S DNA-seq:  The 16S rRNA V3–V4 region was amplified using Tru357F (5’-CGCTCTTCCGATCTCTG TACGGRAGGCAGCAG-3’) and Tru806R (5’-CGCTCTTCCGATCTGAC GGACTACHVGGGTWTCTAAT-3’) primers and sequenced on Illumina MiSeq.

treatment_protocol: Subsequently, mice were shifted to the single-housing (SH) condition (one mouse in one cage of 18 cm length × 11 cm width × 11 cm depth, 198 cm2 floor area) or in the group-housing (GH) condition (same as the acclimatization) with free access to the chow diet and water until PD44. Mice and cecal samples in the different conditions were handled separately under sterile conditions. Cecal content was collected on the PD44, and the mice were euthanized by cervical dislocation. The amygdala, hypothalamus, and liver were collected and stored at −80 °C.

value: Grouip housing

value: Liver

tag: library construction


title: Liver GH4:318G

value: Smarter Stranded Total RNA Sample Prep Kit HI Mammalian


title: E8.5_plate2_H01_RNAseq


title: Dt47.day4.AZA.0.1


title: Dt47.day11.AZA.0


title: plate2_F04_L002_AK7737-GH10_RNAseq


title: Dt47.day11.AZA.0.5


title: Liver GH2:327G


title: plate2_F03_L002_AK10266-GH10_RNAseq


title: Liver GH1:317G


title: Dt47.day11.AZA.0.3


title: E8.5_plate2_G12_RNAseq


title: E8.5_plate2_G11_RNAseq


title: Liver SH3:324S

value: Single housing


title: DMSO.day6.AZA.0

value: DMSO


title: Liver SH4:320S


title: Dt47.day11.AZA.0.1


title: E8.5_plate2_G10_RNAseq


title: plate2_F01_L002_AK5577-GH10_RNAseq


title: plate2_F02_L002_7UDI1240-GH10_RNAseq


title: Liver SH2:334S


title: DMSO.day6.AZA.0.5


title: E8.5_plate2_G07_RNAseq


title: plate2_E12_L002_AK3914-AK4242_RNAseq


title: E8.5_plate2_H02_RNAseq


title: plate2_F06_L002_5UDI232-GH10_RNAseq


title: E8.5_plate2_G08_RNAseq


title: DMSO.day6.AZA.0.3


title: DMSO.day6.AZA.0.1


title: E8.5_plate2_G09_RNAseq


title: Hypothalamus GH3:328G

source_name: Hypothalamus transcriptome

value: Hypothalamus


title: Liver SH1:330S


title: E8.5_plate2_G06_RNAseq


title: plate2_E11_L002_AK16886-AK4242_RNAseq


title: plate2_E10_L002_AK7505-AK4242_RNAseq


title: plate2_E09_L002_AK3910-AK4242_RNAseq


title: Hypothalamus GH4:318G


title: E8.5_plate2_G05_RNAseq


title: Hypothalamus GH1:317G


title: DMSO.day4.AZA.0.1


title: DMSO.day4.AZA.0


title: E8.5_plate2_G03_RNAseq


title: Hypothalamus GH2:327G


title: plate2_E08_L002_AK2870-AK4242_RNAseq


title: DMSO.day4.AZA.0.5


title: plate2_E04_L002_AK7737-AK4242_RNAseq


title: E8.5_plate2_G04_RNAseq


title: plate2_E06_L002_5UDI232-AK4242_RNAseq


title: Hypothalamus SH3:324S


title: Hypothalamus SH4:320S


title: DMSO.day11.AZA.0.3


title: E8.5_plate2_H03_RNAseq


title: E8.5_plate2_F12_RNAseq


title: plate2_E07_L002_AK9176-AK4242_RNAseq


title: DMSO.day4.AZA.0.3


title: E8.5_plate2_G02_RNAseq


title: Amygdala GH4:318G

source_name: Amygdala transcriptome

value: Amygdala


title: DMSO.day11.AZA.0


title: Hypothalamus SH2:334S


title: plate2_E01_L002_AK5577-AK4242_RNAseq


title: plate2_E05_L002_AK9173-AK4242_RNAseq


title: Amygdala GH3:328G


title: E8.5_plate2_G01_RNAseq


title: DMSO.day11.AZA.0.5


title: plate2_D12_L002_AK3914-AK4241_RNAseq


title: plate2_E03_L002_AK10266-AK4242_RNAseq


title: Hypothalamus SH1:330S


title: E8.5_plate2_F11_RNAseq


title: Amygdala GH2:327G


title: B9_Washout_d5

data_processing:
Raw sequences in FASTQ format were mapped to the spike-in Drosophila genome (dm6) using Bowtie2 (version 2.2.9), with those reads not mapped to the spike genome as input to the primary genome (hg19) using Bowtie2 (version 2.2.9).  Mapped reads were then sorted and indexed using samtools (version 1.9). Since Tn5 transposase binds as a dimer and inserts two adaptors separated by 9 bp, all reads mapped to the forward strand need to be offset by +4 bp and those mapped to the reverse strand need to be offset by -5 bp. We used the alignmentSieve command (“--ATACshift”) from deeptools (version 3.5.1) to accomplish the shifting step. Duplicate reads, as well as reads mapped to the human blacklisted regions9, were filtered out. The resulting reads were then used to generate the bigwig files for visualization in genome browser using the bamCoverage command with the CPM (Count Per Million) normalization method.
For each sample, peaks were identified using MACS210 (version 2.1.1), with the narrow peak mode and q value 0.05 as the statistical cutoff. We then pooled the peak summits from all samples and created a non-redundant peak set for further analysis, by the following steps. First, the peak summits pooled from all samples were sorted in descending order by the fifth column (i.e. -log10 P-value). Then we picked the peak summit from the top of the list and removed any remaining peaks within 300 bp of the top peak summit. Then, the next peak summit in the list was picked and the same process was repeated until the entire list was traversed. In total, 156282 non-redundant peak summits were identified. Each peak summit was then extended by 300bp in both directions and the resulting region was used as the peak region.
Genome_build: hg19
Supplementary_files_format_and_content: Bigwig files were generated for visualization in genome browser.  Values represent read count per million (CPM).

extract_protocol:
Omni-ATAC was performed as before (Liu et al. 2021) with 70,000 HUDEP-2 cells frozen in 10% DMSO/FBS. On the day of the experiment, cells were thawed on ice and permeabilized with 50 μL RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) with 0.1% NP40, 0.1% Tween-20 and 0.01% Digitonin for 3 minutes on ice. 1 mL of cold RSB containing 0.1% Tween-20 was added before cells were pelleted again. The pellets were then resuspended with 50 μL transposition mixture (25 μL 2xTD buffer, 2.5 μL transposase from ATAC-seq kit (Illumina), 16.5 μL PBS, 0.5 μL 1% digitonin, and 0.5 μL 10% Tween20), and the reaction was carried out at 37 °C for 45 minutes. Genomic DNA was extracted with a Qiagen MinElute PCR purification kit according to the manufacturer’s recommended protocol.
Total DNA was used as template for amplification, mixed with 15 μL of 2x Ultra II Q5 mix, 1 μL of 10 μM i7 primer (AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG), and 1 μL of 10 μM i5 primer (CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGGAGATGT) where the Ns represent barcodes. PCR amplification was performed as follows: 65°C 5 min, 98°C 30 s, 98°C 10 s, 63°C 30 s, repeat step 3-4 for 5-9x, and 63°C 5min. PCR products were purified with AMPure beads and quantified using Qubit and Tapestation before pooling and sequencing. The libraries were sequenced on the NextSeq 500 platform with NextSeq 500/550 High Output Kit v2 (75 cycles). Paired-end sequencing was performed (2x42 bp, 6 bp index).

treatment_protocol: Cells were resuspended in medium containing 0.15uM dTAG-47. Every other day, cells were centrifuged at 500g for 5min at 4C and resuspended in fresh media containing the drug.

value: immortalized human erythroid progenitor cells

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, ATAC-seq was performed 24h after drug wash out.


title: E8.5_plate2_F10_RNAseq


title: B9_Washout_d8

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, ATAC-seq was performed 4 days after drug wash out.


title: DMSO.day11.AZA.0.1


title: plate2_E02_L002_7UDI1240-AK4242_RNAseq


title: B9_Washout_d4.5

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, ATAC-seq was performed 12h after drug wash out.


title: Amygdala GH1:317G


title: E8.5_plate2_F06_RNAseq


title: B9_Washout_d6

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, ATAC-seq was performed 48h after drug wash out.


title: Amygdala SH4:320S


title: E8.5_plate2_F07_RNAseq


title: B9_dTAG47_d8

value: treated with dTAG47 by 8 days


title: plate2_D09_L002_AK3910-AK4241_RNAseq

value: E7.5 embryo


title: E8.5_plate2_F09_RNAseq

value: E8.5 embryo


title: Amygdala SH3:324S


title: B9_DMSO_d8

value: treated with DMSO by 8 days


title: Amygdala SH2:334S


title: E8.5_plate2_F05_RNAseq


title: E8.5_plate2_F04_RNAseq


title: plate2_D07_L002_AK9176-AK4241_RNAseq


title: Amygdala SH1:330S


title: plate2_D11_L002_AK16886-AK4241_RNAseq


title: plate2_D06_L002_5UDI232-AK4241_RNAseq


title: E8.5_plate2_F03_RNAseq


title: B9_dTAG47_d6

value: treated with dTAG47 by 6 days


title: B9_DMSO_d6

value: treated with DMSO by 6 days


title: plate2_D10_L002_AK7505-AK4241_RNAseq


title: E8.5_plate2_F02_RNAseq


title: RNA-seq ADAR-KDl (shRNA-ADAR) replicate1

city: San Diego

first: Yajing

last: Hao

state: CA

address: George Palade Laboratories 9500 Gilman Drive, Room 217

department: Dept. of Cellular and Molecular Medicine

institute: University of California, San Diego

data_processing:
Quality control was performed using Trimmomatic with parameters MINLEN: 38 and SLIDINGWINDOW: 2:20. Filtered reads were mapped to the reference human genome (hg38) using STAR (Dobin et al. 2013) with the parameters: --outFilterScoreMinOverLread 0.1, –outFilterMatchNminOverLread 0.1, --alignIntronMax 1 and --alignEndsType EndToEnd.
After obtained the mapping files, we removed the duplicate reads using Picard (http://broadinstitute.github.io/picard/) with parameters MarkDuplicates and REMOVE_DUPLICATES = true. We calculated the gene’s reads counts using FeatureCounts(Liao et al. 2014) and converted the reads counts to RPKM using edgeR (Robinson et al. 2010) package in R language. DESeq2(Love et al. 2014) were used to obtain the differerential expressed genes between ADAR knock-down and negative control datasets.
Only genes which FoldChange larger than 2 and adjusted P-Value below 0.05 were retained for further analysis. The wig files of each RNA-seq data set from sense strand and antisense strand were obtained through bamCoverage from DeepTools(Ramirez et al. 2016) with the parameter --ignoreDuplicates and --normalizeUsing CPM.
Genome_build: hg38
Supplementary_files_format_and_content: BigWig, RPKM and differential gene expression results

source_name: Glioblastom Stem Cells

growth_protocol: Neurobasal medium supplemented with B27 (Invitrogen), L-glutamine, sodium pyruvate, 1% penicillin/streptomycin, 20 ng/ml basic fibroblast growth factor (bFGF), and 20 ng/ml epidermal growth factor (EGF)

extract_protocol: Each RNA sample was treated with DNase I (Ambion, DNA-free TM kit). Strictly follow protocols from NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina®.

treatment_protocol: GSC was infected with shADAR lenti-virus to deplete ADAR

value: Glioblastom Stem Cells

tag: genotype

value: ADAR knockdown


title: plate2_D05_L002_AK9173-AK4241_RNAseq


title: plate2_D04_L002_AK7737-AK4241_RNAseq


title: E8.5_plate2_F01_RNAseq


title: RNA-seq control (scrambled shRNA) replicate2

treatment_protocol: GSC was infected with shcontrol lenti-virus

value: control


title: B9_dTAG47_d5

value: treated with dTAG47 by 5 days


title: E8.5_plate2_E12_RNAseq


title: plate2_D03_L002_AK10266-AK4241_RNAseq


title: plate2_D08_L002_AK2870-AK4241_RNAseq


title: RNA-seq ADAR-KDl (shRNA-ADAR) replicate2


title: RNA-seq control (scrambled shRNA) replicate1


title: E8.5_plate2_E11_RNAseq


title: E8.5_plate2_E10_RNAseq


title: plate2_D02_L002_7UDI1240-AK4241_RNAseq


title: E8.5_plate2_E09_RNAseq


title: plate2_D01_L002_AK5577-AK4241_RNAseq


title: plate2_C12_L002_AK3914-GA01_RNAseq


title: B9_DMSO_d4.5

value: treated with DMSO by 4.5 days


title: B9_DMSO_d5

value: treated with DMSO by 5 days


title: B9_dTAG47_d2

value: immortalized human erythroid progenitor cells

value: treated with dTAG47 by 48h


title: B9_DMSO_d2

value: treated with DMSO by 48h


title: E8.5_plate2_E07_RNAseq


title: plate2_C10_L002_AK7505-GA01_RNAseq


title: B9_dTAG47_d1

value: treated with dTAG47 by 24h


title: B9_DMSO_d1

value: treated with DMSO by 24h


title: E8.5_plate2_E08_RNAseq


title: plate2_C09_L002_AK3910-GA01_RNAseq


title: plate2_C08_L002_AK2870-GA01_RNAseq


title: B9_dTAG47_4h

value: treated with dtag47 by 4h


title: E8.5_plate2_E05_RNAseq


title: E8.5_plate2_E04_RNAseq


title: plate2_C07_L002_AK9176-GA01_RNAseq

value: E7.5 embryo


title: B9_DMSO_4h

value: treated with DMSO by 4h


title: B9_dTAG47_d4.5

value: treated with dTAG47 by 4.5 days


title: plate2_C06_L002_5UDI232-GA01_RNAseq


title: E8.5_plate2_E03_RNAseq

value: E8.5 embryo


title: plate2_C05_L002_AK9173-GA01_RNAseq


title: E8.5_plate2_E01_RNAseq


title: E7_Washout_d5

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, ATAC-seq was performed 24h after drug wash out.


title: B9_d0

value: no treatment


title: E8.5_plate2_E02_RNAseq


title: E8.5_plate2_D12_RNAseq


title: plate2_C03_L002_AK10266-GA01_RNAseq


title: plate2_C04_L002_AK7737-GA01_RNAseq


title: E7_Washout_d4.5

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, ATAC-seq was performed 12h after drug wash out.


title: E7_Washout_d6

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, ATAC-seq was performed 48h after drug wash out.


title: plate2_C11_L002_AK16886-GA01_RNAseq


title: E8.5_plate2_E06_RNAseq


title: E8.5_plate2_D11_RNAseq


title: plate2_C02_L002_7UDI1240-GA01_RNAseq


title: plate2_C01_L002_AK5577-GA01_RNAseq


title: plate2_B12_L002_AK3914-AK4240_RNAseq


title: E7_DMSO_d8

value: treated with DMSO by 8 days


title: E8.5_plate2_D10_RNAseq


title: E7_dTAG47_d6

value: treated with dTAG47 by 6 days


title: E8.5_plate2_D09_RNAseq


title: E7_Washout_d8

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, ATAC-seq was performed 4 days after drug wash out.


title: plate2_B11_L002_AK16886-AK4240_RNAseq


title: E7_DMSO_d6

value: immortalized human erythroid progenitor cells

value: treated with DMSO by 6 days


title: E8.5_plate2_D07_RNAseq


title: E8.5_plate2_D08_RNAseq


title: plate2_B10_L002_AK7505-AK4240_RNAseq


title: E7_dTAG47_d5

value: treated with dTAG47 by 5 days


title: E8.5_plate2_D06_RNAseq


title: KM12 GNAS KO in-vitro cell line

city: HOUSTON

first: John Paul

last: Shen

state: TX

address: 1515 Holcombe Blvd

department: Gastrointestinal Medical Oncology

institute: The University of Texas MD Anderson Cancer Center

description: Sample 2

data_processing:
Raw BAM/FASTQ files were mapped to the human reference genome (hg38) using STAR (version 2.7.2b) RNASeq alignment tool. Biobambam (version 0.0.191) was used to mark duplicates and index raw files before alignment.
Xenome (version 1.0.1-r) was used to segregate mouse and human reads from raw RNAseq files of CDX models. Mouse reference genome (GRCm39) and gene annotation file (Gencode.vM26) were used for mapping and annotating mouse reads. Extracted human reads for each CDX sample were stored in FASTQ files and input to the downstream STAR (version 2.7.2b) RNASeq alignment tool.
HTSeq (version 0.11.0) was utilized to extract raw read counts from STAR-aligned RNAseq files of both cell lines and CDX models.
Assembly: hg38
Supplementary files format and content: tab-delimited text file include raw counts for each Sample

growth_protocol: All cell lines were grown in RPMI-1640 medium supplemented with 10% FBS, 2mM glutamine, and 1% Pen-Strep at standard culture conditions (37 °C, 5% CO2, 95% humidity). Cells were maintained at low passages, authenticated by STR analysis and tested to rule out mycoplasma contamination.

extract_protocol:
RNA was extracted using RNAqueous™ Total RNA Isolation Kit (Catalog number: AM1912). RNA concentraction ≥ 20 ng/µl for each sample was used for library construction.
Before library construction, quality control for each RNA samples was performed to guarantee the reliability of the data. RNA degradation and potential contamination were tested by Agarose Gel Electrophoresis. Agilent 2100 was used to check the RNA integrity and quantitation. After the QC procedures, mRNA from each smaple was enriched using oligo(dT) beads. First, the mRNA was fragmented randomly by adding fragmentation buffer, then the cDNA was synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I were added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library was completed through size selection and PCR enrichment.

value: Colorectal cancer cell line

value: GNAS Knock-out


title: LS174T GNAS R201H cell lines Dox (-)

description: Sample 5

value: GNASR201H Dox(-)


title: E8.5_plate2_D05_RNAseq


title: E7_DMSO_d5

value: treated with DMSO by 5 days


title: plate2_B09_L002_AK3910-AK4240_RNAseq

value: E7.5 embryo


title: plate2_B08_L002_AK2870-AK4240_RNAseq


title: E8.5_plate2_D04_RNAseq


title: E7_dTAG47_d8

value: treated with dTAG47 by 8 days


title: E7_dTAG47_d4.5

value: treated with dTAG47 by 4.5 days


title: E7_DMSO_d4.5

value: treated with DMSO by 4.5 days


title: LS174T GNAS R201H cell lines Dox (+)

description: Sample 6

value: GNASR201H Dox(+)


title: plate2_B07_L002_AK9176-AK4240_RNAseq


title: E7_dTAG47_d2

value: treated with dTAG47 by 48h


title: plate2_B05_L002_AK9173-AK4240_RNAseq


title: E8.5_plate2_D02_RNAseq

value: E8.5 embryo


title: plate2_B04_L002_AK7737-AK4240_RNAseq


title: plate2_B06_L002_5UDI232-AK4240_RNAseq


title: E8.5_plate2_D01_RNAseq


title: E7_dTAG47_d1

value: treated with dTAG47 by 24h


title: plate2_B03_L002_AK10266-AK4240_RNAseq


title: E7_DMSO_d2

value: treated with DMSO by 48h


title: E7_DMSO_d1

value: treated with DMSO by 24h


title: plate2_B02_L002_7UDI1240-AK4240_RNAseq


title: E7_dTAG47_4h

value: immortalized human erythroid progenitor cells

value: treated with dtag47 by 4h


title: E8.5_plate2_C11_RNAseq


title: KM12 parental in-vitro cell line

description: Sample 1


title: E8.5_plate2_C10_RNAseq


title: E7_DMSO_4h

value: treated with DMSO by 4h


title: plate2_B01_L002_AK5577-AK4240_RNAseq


title: plate2_A12_L002_AK3914-AK4239_RNAseq


title: E8.5_plate2_C08_RNAseq


title: E8.5_plate2_C09_RNAseq


title: E7_d0

value: no treatment


title: E8.5_plate2_D03_RNAseq


title: plate2_A11_L002_AK16886-AK4239_RNAseq


title: E8.5_plate2_C07_RNAseq


title: plate2_A10_L002_AK7505-AK4239_RNAseq


title: plate2_A09_L002_AK3910-AK4239_RNAseq


title: E8.5_plate2_C06_RNAseq


title: E8.5_plate2_C12_RNAseq


title: E8.5_plate2_C05_RNAseq


title: E8.5_plate2_C04_RNAseq


title: plate2_A07_L002_AK9176-AK4239_RNAseq


title: 990_ATAC-seq

city: Chicago

first: Feng

last: Yue

state: IL

address: 303 E Chicago Ave. Simpson Querrey 7-518

department: Biochemistry and Molecular Genetics

institute: Northwestern University

data_processing:
Reads were first trimmed by Cutadapt (version 2.4) with “-m 5 -e 0.2” options
Trimmed reads were then aligned to human genome reference GRCh38 with “bowtie2 -X2000 --mm” options
Mapped reads were deduplicated using Picard MarkDuplicates tool with “VALIDATION_STRINGENCY=LENIENT” option
Read alignments were shifted +4bp on “+” strand and -5bp on “-” strand to account for Tn5 insertion before peak calling.
Peaks were called by MACS2 with “--shift -75  --extsize 150 --nomodel -B --SPMR --keep-dup all --call-summits” options, and filtered against the ENCODE hg38 blacklist.
Genome_build: hg38
Supplementary_files_format_and_content: Peak, ATAC-seq peaks called by MACS2, filter by p value < 1e-5

source_name: Primary AML

extract_protocol:
ATAC-seq was performed following the published protocol with minimal modification[Buenrostro et al., Curr Protoc Mol Biol, 2015]. Briefly, we also centrifuge down 50,000 cells, wash the cells with PBS, and perform nuclei extraction with cold lysis buffer.  We added an extra step of washing the nuclei with another 500ul of lysis buffer to further remove mitochondrial DNA.
We then proceed with the transposition reaction (Illumina Tagment DNA Enzyme and Buffer Large Kit 20034198) and purification steps (QIAGEN MinElute PCR Purification Kit Cat No./ID: 28004) . We elute the DNA in 20ul elution buffer instead of 10ul elution buffer to increase recovery rate. Then we proceed with the PCR amplification step, where we use 20ul transposed DNA, 2.5ul of Nextera PCR primer 1 and 2.5ul of Nexteral primer 2, and 25ul KAPA HiFi HotStart Ready Mix master mix (KAPA KR0370). We use the PCR parameters indicated by the standard protocol with 11 cycles. We then perform size selection to remove small fragments using KAPA pure beads. We add 45ul KAPA beads to 50ul PCR solution, incubate for 15 minutes in room temperature, and use a magnet to capture the beads and discard the supernatant. We wash the beads with 200ul of 80% ethanol twice and remove the ethanol. We resuspend the beads in 20-50ul of prewarmed 10mM Tris-HCl, pH 8.0, incubate at 37 for 10 minutes, and use a magnet to recollect the supernatant as the final library. We use 1ul of the library to run on a 2% agarose gel to verify the footprint nucleosomes for successful assay. Libraries were sequenced as 150bp paired-end reads on platform Hiseq 4000 with 20 million raw read pairs per sample.

value: Selected PBMC or BM cells

tag: assay

value: ATAC-seq


title: plate2_A06_L002_5UDI232-AK4239_RNAseq


title: 168_ATAC-seq


title: E8.5_plate2_C03_RNAseq


title: 1360_ATAC-seq


title: 027_ATAC-seq


title: E8.5_plate2_C02_RNAseq


title: plate2_A05_L002_AK9173-AK4239_RNAseq


title: plate2_A04_L002_AK7737-AK4239_RNAseq


title: 1916_ATAC-seq


title: E8.5_plate2_C01_RNAseq


title: 472_ATAC-seq


title: 546_ATAC-seq


title: E8.5_plate2_F08_RNAseq


title: 260_ATAC-seq


title: plate2_A03_L002_AK10266-AK4239_RNAseq


title: A172_Stable_RBFOX1_05_3

city: Montreal

first: Gabrielle

last: Perron

state: Quebec

address: 845 Sherbrooke St W

department: Human Genetics

country: Canada

institute: McGill University

description: A172 stable cell line expressing RBFOX1

data_processing:
RNA-seq reads were aligned to the GRCh38 genome assembly using HISAT2
RNA-seq reads to the human genome (GRCh38 assembly, Ensembl release 85) were counted using htseq-count, with a MAPQ score cutoff of >= 30
Differentially expressed genes were identified using DESeq2
Assembly: GRCh38
Supplementary files format and content: tab-delimited text files contain raw counts for each sample

source_name: Glioblastoma cell line

growth_protocol: The human glioblastoma cell line A-172 was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C with 5% CO2.

extract_protocol:
Total RNA was extracted using All Prep DNA/RNA/miRNA Universal kit (Qiagen)
Library preparation was performed using NEB rRNA-depleted (HMR) stranded library preparation kit accorind to manufacturer's instructions.

treatment_protocol: HEK293T cells were transfected with lentiviral packaging plasmids (psPAX2 and pMD2.G.) together with a lentiviral expression plasmid (GFP or RBFOX1) using Lipofectamine 3000. After 48 h, medium containing lentiviral particles was collected, filtered using a 0.45 μm syringe filter, and immediately added to A-172 cells with 8 μg/ml polybrene.

value: overexpressing RBFOX1


title: plate2_A08_L002_AK2870-AK4239_RNAseq

value: E7.5 embryo


title: A172_Stable_RBFOX1_05_2

description: A172 stable cell line expressing RBFOX1


title: E8.5_plate2_B11_RNAseq


title: plate2_A01_L002_AK5577-AK4239_RNAseq


title: 773_ATAC-seq


title: E8.5_plate2_B12_RNAseq


title: 018_ATAC-seq


title: plate2_A02_L002_7UDI1240-AK4239_RNAseq


title: E8.5_plate2_B09_RNAseq


title: A172_Stable_RBFOX1_05_1

description: A172 stable cell line expressing RBFOX1


title: plate1_H12_L001_AK6249-AK4238_RNAseq


title: 270_ATAC-seq


title: E8.5_plate2_B10_RNAseq


title: E8.5_plate2_B08_RNAseq


title: A172_Stable_GFP_05_3

description: A172 stable control cell line expressing GFP

value: overexpressing GFP


title: A172_Stable_GFP_05_2


title: 629_ATAC-seq


title: 566_ATAC-seq


title: plate1_H10_L001_AK9404-AK4238_RNAseq


title: 103_ATAC-seq

source_name: Primary Healthy donor


title: 576_ATAC-seq


title: plate1_H07_L001_AK8571-AK4238_RNAseq


title: siSCR Replicate3

city: Hull

first: Mark

last: Wade

state: Humberside

address: Room 121, Hardy Building, University of Hull, Cottingham Road

institute: University of Hull

description: Sample 5

data_processing:
Original image data file is transformed to sequence reads by CASAVA base recognition.
Raw reads were filtered to remove reads containing adapters or reads of low quality. Filtering process: 1) Discard reads with adapter contamination. 2) Discard reads when uncertain nucleotides constitute more than 10 percents of either read (N > 10%). 3) Discard reads when low quality nucleotides (base quality less than 20) constitute more than 50 percents of the read.
Read alignment performed on filtered reads using the HISAT2 algorithm and read counts and FPKM calculated for each gene using HTSeq. Normalised readcounts were used for subsequent differential gene expression analysis using the DESeq2 R package.
Assembly: Genome Reference Consortium Human Build 38
Supplementary files format and content: Matrix table with FPKM values for every gene and every sample

growth_protocol: Cells were cultured in RPMI-1640 media (Gibco) containing 10% foetal-calf serum (FCS) (Gibco) and 1% penicillin/streptomycin (Merck).

extract_protocol:
Total RNA was extracted using the Rneasy Mini Kit (Qiagen) according to manufactorers instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: Cell lines were transfected using Lipofectamine RNAiMAX (Invitrogen) with siRNA to a final concentration of 25 nM, according to manufacturer recommendations and grown for 72 hours prior to RNA extraction.

value: siSCR


title: siCBX2#3 Replicate2

description: Sample 4


title: plate1_H08_L001_AK7085-AK4238_RNAseq


title: E8.5_plate2_B01_RNAseq


title: plate1_H05_L001_5UDI219-AK4238_RNAseq


title: siCBX2#3 Replicate3

description: Sample 6


title: E8.5_plate2_B05_RNAseq


title: MB002_shMSI_3_poly

city: La Jolla

first: En-Ching

last: Luo

address: 9500 Gilman St

institute: UCSD

description:
MSI3_poly
processed data file: msi_polysome_RPKM.csv

data_processing:
RNA-sequencing reads were trimmed using cutadapt (v1.4.0) of adaptor sequences, and mapped to repetitive elements (RepBase v18.04) using the STAR (v2.4.0i). Reads did not map to repetitive elements were then mapped to the human genome (hg19). Using GENCODE (v19) gene annotations and featureCounts (v.1.5.0) to create read count matrices.
The transcript RPKMs of input and polysome fractions were calculated from the read count matrices.
Genome_build: Homo sapiens UCSC hg19 (GRCh37)
Supplementary_files_format_and_content: msi_polysome_RPKM.csv: Comma-separated text file includes RPKM values.

source_name: patient-derived medulloblastoma cell

growth_protocol: Cells (MB002) were grown in BTIC enrichment media until 20 million cells were cultured.

extract_protocol:
The total RNA is then extracted from the remaining lysate using Trizol LS (Invitrogen). The polysome fractions are collected and pooled for polyribosome associated RNA extraction using Trizol LS. Extracted polysome associated RNA is resuspended in 50μL of sterile ultrapure water.
1ug of total RNA and an equivalent volume of RNA from the polysome fraction were used for library preparation as per Illumina standard RNA-seq protocol. An equal amount of Spike ins RNA Variant Controls (SIRVs, Lexogen SIRV-Set3 and ERCC) were added to each sample prior to library preparation to normalize for sequencing bias and to determine the threshold for measurable statistics. The library was then used to build clusters on the Illumina flow cell and analysis was done using Illumina Hiseq 4000 platform (Illumina, San Diego, CA, USA) at the UCSD Institute for Genomic Medicine to a target depth of 30M reads per sample.

treatment_protocol: 20M cells incubated for 10min with 0.1mg of cycloheximide (Sigma)/mL of BTIC media and incubated for 5min at 37oC and 5% CO2. Cells were then washed and harvested in ice cold PBS and flash frozen in liquid nitrogen and stored until ready to lyse pellets. Polysome buffer (20mM Tris HCl pH7.4, 150mM NaCl, 5mM MgCl2, 1mM DTT) and lysis buffer (Polysome Buffer with 1% Triton-X + Protease Inhibitors + RNase inhibitors + cycloheximide (100μg/ml)) were prepared fresh and kept on ice. Pellets were lysed in 400μL of Lysis Buffer per 10x10e6 cells. Lysate is then incubated on ice for 30 min then centrifuged at 15,000 rpm at 4oC for 2 minutes to remove debris. The supernatant is collected and loaded onto a 10-50% sucrose gradient in Polysome buffer, and centrifuged at 35,000 x g (Beckman, rotor SW41) at 4 °C for 3 h. Fractions were collected from the top and UV absorbance monitored using a Gradient Station (BioCamp) equipped with ECONO UV monitor (BioRad). 500 µl fraction were collected using a FC203B (Gilson) fraction collector, and total polysome were pooled.

tag: pdx line

tag: shRNA

value: shMSI

tag: rna fraction

value: Polysome


title: plate1_H04_L001_AK840-AK4238_RNAseq


title: E8.5_plate2_B02_RNAseq


title: MB002_shMSI_2_poly

description:
MSI2_poly
processed data file: msi_polysome_RPKM.csv


title: siCBX2#3 Replicate1

description: Sample 2


title: plate1_H03_L001_5UDI265-AK4238_RNAseq


title: plate1_H11_L001_AK9170-AK4238_RNAseq


title: E8.5_plate2_B06_RNAseq


title: E8.5_plate2_A12_RNAseq


title: 673_ATAC-seq


title: E8.5_plate2_B04_RNAseq


title: E8.5_plate2_B07_RNAseq


title: siSCR Replicate1

description: Sample 1


title: MB002_shMSI_2

description:
MSI2
processed data file: msi_polysome_RPKM.csv

value: Total


title: A172_Stable_GFP_05_1


title: MB002_GFP_3_poly

description:
GFP3_poly
processed data file: msi_polysome_RPKM.csv

value: GFP


title: plate1_H09_L001_AK6296-AK4238_RNAseq


title: plate1_H06_L001_AK763-AK4238_RNAseq


title: E8.5_plate2_B03_RNAseq

value: E8.5 embryo


title: MB002_shMSI_3

description:
MSI3
processed data file: msi_polysome_RPKM.csv


title: siSCR Replicate2

description: Sample 3


title: siCBX2#3 Replicate_3

description: Sample 9


title: MB002_GFP_3

description:
GFP3
processed data file: msi_polysome_RPKM.csv


title: plate1_H02_L001_AK5426-AK4238_RNAseq


title: siCBX2#2 Replicate_3

description: Sample 8


title: E8.5_plate2_A10_RNAseq


title: E8.5_plate2_A11_RNAseq


title: MB002_GFP_2_poly

description:
GFP2_poly
processed data file: msi_polysome_RPKM.csv


title: plate1_H01_L001_AK2831-AK4238_RNAseq


title: siSCR Replicate_3

description: Sample 7


title: E8.5_plate2_A09_RNAseq


title: siCBX2#2 Replicate_2

description: Sample 5


title: siCBX2#3 Replicate_2

description: Sample 6


title: siSCR Replicate_2

description: Sample 4


title: plate1_G10_L001_AK9404-7UDI250_RNAseq


title: plate1_G12_L001_AK6249-7UDI250_RNAseq


title: plate1_G11_L001_AK9170-7UDI250_RNAseq


title: E8.5_plate2_A07_RNAseq


title: E8.5_plate2_A08_RNAseq


title: plate1_G09_L001_AK6296-7UDI250_RNAseq


title: plate1_G08_L001_AK7085-7UDI250_RNAseq


title: siCBX2#3 Replicate_1

description: Sample 3


title: Healthy mucosa of non-IBD control #7

city: Seoul

first: Sung Noh

last: Hong

state: South Korea

address: 81 Irwon-Ro, Gangnam-gu

department: Gastroenterolgy

country: South Korea

institute: Samsung Medical Center

data_processing:
Several steps including a reverse transcription reaction with poly-(dT) primers using SuperScriptTM II reverse transcriptase (Invitrogen/Life Technologies, Grand Island, NY, USA), cDNA amplification, end-repair, 3′-end adenylation, adapter ligation, and amplification were conducted.
The quantity and quality of the library were measured using a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a 4200 TapeStation instrument (Agilent Technologies, Santa Clara, CA, USA).
Sequencing of the transcriptome library was carried out using the 100 bp paired-end mode of a TruSeq Rapid PE Cluster kit and a TruSeq Rapid SBS kit (Illumina) on a HiSeq 2500 sequencing platform (Illumina).
Sequence reads as FASTQ files were mapped against the human reference genome build 19 (hg19) using TopHat v.2.0.6 (http://ccb.jhu.edu/software/tophat/index.shtml).
Raw read counts mapped to genes were measured using the BAM format file with HTSeq version 0.6.1 (https://htseq.readthedocs.io).
Assembly: hg19
Supplementary files format and content: tab-delimited text file includes raw read counts for each sample

source_name: Healthy mucosa

growth_protocol: Biopsy specimens were immediately frozen in liquid nitrogen and stored at -80 °C.

extract_protocol:
Total RNA was extracted by combining 2-4 biopsy samples obtained using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions.
Library construction was performed using a TruSeq RNA Sample Prep Kit v.2 (Illumina, San Diego, California, USA).

treatment_protocol:
N/A
N/A

tag: disease state

value: control

tag: individual id

value: control #7

value: Healthy mucosa


title: Healthy mucosa of non-IBD control #9

value: control #9


title: Healthy mucosa of non-IBD control #8

value: control #8


title: Healthy mucosa of non-IBD control #6

value: control #6


title: siCBX2#2 Replicate_1

description: Sample 2


title: plate1_G07_L001_AK8571-7UDI250_RNAseq


title: E8.5_plate2_A04_RNAseq


title: E8.5_plate2_A05_RNAseq


title: MB002_GFP_2

description:
GFP2
processed data file: msi_polysome_RPKM.csv


title: plate1_G06_L001_AK763-7UDI250_RNAseq


title: siSCR Replicate_1

description: Sample 1


title: Healthy mucosa of non-IBD control #4

value: control #4


title: E8.5_plate2_A02_RNAseq


title: E8.5_plate2_A03_RNAseq


title: Healthy mucosa of non-IBD control #5

value: control #5


title: plate1_G05_L001_5UDI219-7UDI250_RNAseq


title: Healthy mucosa of non-IBD control #2

value: control #2


title: Healthy mucosa of non-IBD control #3

value: control #3


title: E8.5_plate2_A06_RNAseq


title: plate1_G03_L001_5UDI265-7UDI250_RNAseq

value: E7.5 embryo


title: plate1_G04_L001_AK840-7UDI250_RNAseq


title: E8.5_plate2_A01_RNAseq


title: E8.5_plate1_H12_RNAseq


title: haec, Input

city: Tianjin

first: Xi

last: Wang

address: Qixiangtai Road, No. 12

department: The School and Hospital of Stomatology

country: China

institute: Tianjin Medical University

data_processing:
Raw sequence reads were initially processed by FASTQC for quality control, and then adapter sequences and poor quality reads were removed by using Trimmomatic.Quality filtered reads were then mapped to hg38 genome using STAR.
To minimize the rate of false positives, only uniquely mapped and withoutduplication reads were selected by samtools and were separated by strand for those strand-specific samples with RSeQC.
To facilitate the reads coverage visualization and comparison among different samples,UCSC tools and RSeQC were employed for bigwig format transforming and normalization separately.
m6A site were detected using transcriptomebased peak caller MeTPeak, and the top 5,000 peaks were chosen for denovo motif analysis with DREME, which takes 200-nt-long peak summit-centered sense sequences as input.
Obtained the annotation of m6a sites on the whole genome using ChIPseeker.
Merged the peak files of different group using merge in bedtools toolset.
Calculated the signal in two different groups on every merged m6A peaks with UCSC tools.
Genome_build: hg38
Supplementary_files_format_and_content: bw files were generated using to m6A-specific Peak sequence Motif analysis.

source_name: human aortic endothelial cells (HAEC)

growth_protocol: Total RNA from SCC25 and HAEC in culture was extracted using Trizol reagent (15596018; Thermo Fisher Scientific, Waltham, MA). Due to the presence of m6A in DNA, total RNA was treated with DNase I (04 716 728 001;Roche Diagnostics, Indianapolis, IN) for 20 minutes at 37 C to remove DNA contamination. The RNA was precipitated using glycogen (25 μg/mL final) (5 mg/mL; AM9510; Thermo Fisher Scientific) and isopropanol at -30 C for 2 hours. The precipitated RNA was then washed with 70% ethanol. The final pellet was resuspended in ultrapure H2O. The concentration of total RNA was measured by Qubit RNA HS Assay Kit (Q32855; Thermo Fisher Scientific).

extract_protocol:
m6A MeRIP is based on the previously described m6A-seq protocol[1] with several modifications: 30 μl of protein A magnetic beads (10002D; Thermo Fisher Scientific) and 30 μl of protein G magnetic beads (10004D; Thermo Fisher Scientific) were washed twice by IP buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), resuspended in 500 μl of IP buffer, and tumbled with 5 μg anti-m6A antibody at 4 C for at least 6 hours. Following 2 washes in IP buffer, the antibody-bead mixture was resuspended in 500 μl of the IP reaction mixture containing fragmented total RNA, 100 μl of 5× IP buffer, and 5 μl of RNasin Plus RNase Inhibitor (N2611; Promega, Madison, WI) and incubated for 2 hours at 4 C. In the low/high salt-washing method, the RNA reaction mixture was washed twice in 1,000 μl of IP buffer, twice in 1,000 μl of low-salt IP buffer (50 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.1% IGEPAL CA-630 in nuclease-free H2O), and twice in 1,000 μl of high-salt IP buffer (500 mM NaCl, 10 mM Tris-HCl [pH 7.5],0.1% IGEPAL CA-630 in nuclease-free H2O) for 10 minutes each at 4 C. After extensive washing, the m6Aenriched fragmented. RNA was eluted from the beads in 200 μl of RLT buffer supplied in RNeasy Mini Kit (74106; QIAGEN; Germany) for 2 minutes at room temperature. A magnetic separation rack was used to pull beads to the side of the tube. Supernatant was collected to a new tube, and 400 μl of 100% ethanol was added to it. The mixture was transferred to an RNeasy MiniElute spin column (QIAGEN) and centrifuged at >12,000 rpm at 4 C for 1 minute. The spin column membrane was washed with 500 μl of RPE buffer once, then 500 μl of 80% ethanol once, and centrifuged at full speed for 5 minutes at 4 C to remove the residual ethanol. The m6A-enriched RNA was eluted with 14 μl ultrapure H2O.
The amount of 2 μl of 14 μl eluted RNA was reverse transcribed with High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Once successfully immunoprecipitated methylated RNA was confirmed, further transcriptome-wide interrogation was pursued by deep sequencing using SMARTer Stranded Total RNA-Seq Kit version 2 (Pico Input Mammalian; 634413; Takara/Clontech, Japan) according to the manufacturer's protocol. A purified library was quantified using a Qubit Fluorometer (Thermo Fisher Scientific), and the size distribution was checked using the Bioanalyzer 2100 system (Agilent Technologies). The library preparations were sequenced on an Illumina Novaseq 6000 platform and 150 bp paired-end reads were generated.
MeRIP-seq

treatment_protocol: RNA fragmentation is based on the previously described m6A-seq protocol [1,2] with a few modifications: the total volume of approximately 3 to 5 μg total RNA was adjusted to 18 μl with RNase-free water. The amount of 2 μl of 10X RNA Fragmentation Buffer (100 mM Tris- HCl, 100 mM ZnCl2 in nuclease-free H2O) was added and incubated in a preheated thermal cycler for approximately 5 to 6 minutes at 70 C. The reaction was stopped by adding 2 μl of 0.5M EDTA. Then added to the mixture was 178 μl of H2O, 20 μl of sodium acetate (3M [pH 5.2]; S7899; Sigma-Aldrich, St. Louis, MO), 14.4 μl of glycogen (5 mg/mL; AM9510; Thermo Fisher Scientific), and 500 μl of 100% ethanol, and the mixture was incubated at -80 C overnight. Fragmented RNA was pelleted by centrifuge, washed once with 75% ethanol, and resuspended in ultrapure H2O (10 μl H2O per 1 μg human total RNA). The size distribution of fragmented RNA was assessed using Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The total RNA was chemically fragmented into approximately 200-nt-long fragments.

tag: cell phenotype

value: anchorage-dependent cell

value: human aortic endothelial cells

tag: rip antibody


title: scc25, Input

source_name: human tongue phosphorus carcinoma cells

value: human tongue squamous cell carcinoma cells


title: scc25, IP

value: anti-m6A, E1610S(NEB)


title: Healthy mucosa of non-IBD control #1

value: control #1


title: haec, IP


title: E8.5_plate1_H10_RNAseq

tag: strain

value: E8.5 embryo


title: E8.5_plate1_H11_RNAseq

tag: tissue


title: plate1_G01_L001_AK2831-7UDI250_RNAseq


title: plate1_G02_L001_AK5426-7UDI250_RNAseq


title: non-inflamed mucosa of CD #13

source_name: non-inflamed mucosa

value: Crohn's disease

value: non-inflamed mucosa


title: Inflamed mucosa of CD #13

source_name: Inflamed mucosa

value: Inflamed mucosa


title: E8.5_plate1_H09_RNAseq


title: HUDEP2_B9_Washout_d8

data_processing:
Library strategy: PRO-seq
Raw paired-end sequences in FASTQ format were trimmed using cutadapt1 (version 1.18) to remove adapter sequences and low-quality 3’ bases (--match-read-wildcards -m 20 -q 10). The 3’ adapter sequences of R1 reads (“TGGAATTCTCGGGTGCCAAGGAACTCCAGTCAC”) and the 3’ adapter of R2 reads (“GATCGTCGGACTGTAGAACTCTGAAC”) were removed from all read pairs. The resulting read pairs were mapped to the spike-in Drosophila genome (dm6) using Bowtie22 (version 2.2.9), with those reads not mapped to the spike genome as input to the primary genome (hg19) using Bowtie2. Reads mapped to the hg19 reference genome were then sorted and indexed using samtools3 (version 1.9), and subsequently converted to BED format using the bamtobed command from the bedtools suite4 (version 2.30.0). The 3’ end positions of the R1 end, which correspond to the 3’ end of nascent RNAs, were extracted from the BED files and used as input to generate bedGraph files at single-nucleotide resolution for visualization using the genomecov command from bedtools, with a genome-wide normalization factor being 30 million divided by total read numbers. Thus the signals in the bedGraph files represent the number of nascent transcripts per 30 million transcripts. Because the R1 end in PRO-seq reveals the position of the RNA 3’ end, the “+” and “-“ strands were swapped to generate bedGraphs representing 3’ end position at single-nucleotide resolution.
Genome_build: hg19
Supplementary_files_format_and_content: BedGraph files at single-nucleotide resolution were generated for visualization in genome browsers. Nascent transcription activities at the forward and reverse strands are merged into one single file. Values represent Read Counts per 30 million nascent transcripts.

extract_protocol:
PRO-seq was performed as described (Liu et al. 2021). Five million HUDEP-2 were harvested on ice, and washed with cold PBS. Cells were resuspended in 2 mL cold Buffer W (10 mM Tris-Cl, pH 8.0, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 0.5 mM DTT, 10% glycerol, 0.5% BSA) using wide-bore P1000 tip and transferred to a 2 mL low binding tube and strained through a 35 micron nylon mesh cell strainer. Immediately add 18 mL Chilled Buffer P (Buffer W supplemented with 0.05% Igepal CA630) to cell suspension and mix gently for 2 min. Cells were centrifuged at 700 x g for 8 min and resuspended in 1 mL Buffer F (50 mM Tris-HCl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.5 mM DTT, 0.5% BSA), and centrifuged again at 700 x g for 8 min. The pellets were resuspended in 400 μL Buffer F and counted using a hemocytometer to ensure that >95% cells were permeabilized. The cell density was adjusted to 1 x 106/100 μL, and the suspension was snap-frozen and stored at -80°C.
cDNA was immediately amplified for 5-cycles ("preCR" step).  A PCR cocktail consisting of NEBNext Ultra II Q5 master mix (NEB) and Illumina TruSeq PCR primers (RP-1, common; and RPI-X, indexing) was added to each cDNA and amplification carried out according to manufacturer's suggested 2-step cycling conditions for NGS applications. To determine optimal library amplification, the preCRs were thawed on ice and an aliquot serially diluted. Each dilution was mixed with a PCR cocktail (Q5 DNA polymerase with optional high GC enhancer) containing 10 mM/ea of Illumina universal P5 and P7 primers and amplified 15-cycles.  The reactions were electrophoresed on a 2.2% agarose gel (SeaKem) cast with SYBR gold (Thermo) and analyzed to determine the optimal number of cycles for final amplification. preCRs were transferred from ice to a pre-heated thermal cycler block and amplified for the appropriate number of additional cycles to reach the total determined in the test amplification. Pooled libraries were sequenced using an Illumina NovaSeq S1 (PE 50), Novaseq S4 (200 cycle kit), NextSeq in high output mode (PE 75), NovaSeq S1 with 100 cycle kit (PE50).

treatment_protocol: Cells were resuspended in medium containing 0.15uM dTAG-47. Every other day, cells were centrifuged at 500g for 5min at 4C and resuspended in fresh media containing the drug. After 4 days in drug, cells were washed with PBS and put in media devoid of dTAG-47.

tag: cell-type

value: immortalized human erythroid progenitor cells

tag: cell line

tag: rna source

value: nuclear run-on

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, PRO-seq was performed 4 days after drug wash out.


title: plate1_F12_L001_AK6249-7UDI263_RNAseq


title: non-inflamed mucosa of CD #12


title: HUDEP2_B9_DMSO_d8

tag: treatment

value: treated with DMSO by 8 days


title: Inflamed mucosa of CD #12


title: non-inflamed mucosa of CD #11


title: plate1_F11_L001_AK9170-7UDI263_RNAseq


title: plate1_F10_L001_AK9404-7UDI263_RNAseq


title: E8.5_plate1_H08_RNAseq


title: E8.5_plate1_H06_RNAseq


title: Inflamed mucosa of CD #11


title: E8.5_plate1_H07_RNAseq


title: HUDEP2_B9_dTAG47_d6

value: treated with dTAG47 by 6 days


title: plate1_F09_L001_AK6296-7UDI263_RNAseq


title: plate1_F08_L001_AK7085-7UDI263_RNAseq


title: HUDEP2_B9_Washout_d5

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, PRO-seq was performed 24h after drug wash out.


title: HUDEP2_B9_DMSO_d6

value: treated with DMSO by 6 days


title: E8.5_plate1_H04_RNAseq


title: E8.5_plate1_H03_RNAseq


title: plate1_F07_L001_AK8571-7UDI263_RNAseq

value: E7.5 embryo


title: HUDEP2_B9_dTAG47_d8

value: treated with dTAG47 by 8 days


title: non-inflamed mucosa of CD #9

value: CD #9


title: Inflamed mucosa of CD #10


title: HUDEP2_B9_dTAG47_d5

value: treated with dTAG47 by 5 days


title: HUDEP2_B9_DMSO_d5

value: treated with DMSO by 5 days


title: Inflamed mucosa of CD #9


title: plate1_F06_L001_AK763-7UDI263_RNAseq


title: plate1_F05_L001_5UDI219-7UDI263_RNAseq


title: HUDEP2_B9_Washout_d4.5

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, PRO-seq was performed 12h after drug wash out.


title: HUDEP2_B9_Washout_d6

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, PRO-seq was performed 48h after drug wash out.


title: E8.5_plate1_H01_RNAseq

value: E8.5 embryo


title: E8.5_plate1_H02_RNAseq


title: non-inflamed mucosa of CD #8

value: Crohn's disease

value: CD #8

value: non-inflamed mucosa


title: plate1_F04_L001_AK840-7UDI263_RNAseq


title: non-inflamed mucosa of CD #10


title: HUDEP2_B9_dTAG47_d4.5

value: immortalized human erythroid progenitor cells

value: nuclear run-on

value: treated with dTAG47 by 4.5 days


title: E8.5_plate1_H05_RNAseq


title: plate1_F03_L001_5UDI265-7UDI263_RNAseq


title: Inflamed mucosa of CD #8

value: Inflamed mucosa


title: E8.5_plate1_G11_RNAseq


title: non-inflamed mucosa of CD #7

value: CD #7


title: HUDEP2_B9_DMSO_d4.5

value: treated with DMSO by 4.5 days


title: plate1_F02_L001_AK5426-7UDI263_RNAseq

value: E7.5 embryo


title: plate1_F01_L001_AK2831-7UDI263_RNAseq


title: Inflamed mucosa of CD #7


title: E8.5_plate1_G09_RNAseq


title: E8.5_plate1_G10_RNAseq


title: plate1_E12_L001_AK6249-AK4236_RNAseq


title: non-inflamed mucosa of CD #6

value: CD #6


title: E8.5_plate1_G08_RNAseq


title: HUDEP2_E7_DMSO_d8

value: treated with DMSO by 8 days


title: HUDEP2_E7_dTAG47_d8

value: treated with dTAG47 by 8 days


title: Inflamed mucosa of CD #6


title: HUDEP2_E7_Washout_d8

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, PRO-seq was performed 4 days after drug wash out.


title: plate1_E11_L001_AK9170-AK4236_RNAseq


title: HUDEP2_E7_Washout_d6


title: E8.5_plate1_G12_RNAseq


title: non-inflamed mucosa of CD #5

value: CD #5


title: E8.5_plate1_G07_RNAseq


title: E8.5_plate1_G06_RNAseq


title: E8.5_plate1_G05_RNAseq


title: Inflamed mucosa of CD #5


title: non-inflamed mucosa of CD #4

value: CD #4


title: plate1_E08_L001_AK7085-AK4236_RNAseq


title: plate1_E10_L001_AK9404-AK4236_RNAseq


title: HUDEP2_E7_DMSO_d6

value: treated with DMSO by 6 days


title: E8.5_plate1_G04_RNAseq

value: E8.5 embryo


title: plate1_E09_L001_AK6296-AK4236_RNAseq


title: HUDEP2_E7_Washout_d5

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, PRO-seq was performed 24h after drug wash out.


title: Inflamed mucosa of CD #4

value: Crohn's disease


title: plate1_E07_L001_AK8571-AK4236_RNAseq


title: E8.5_plate1_G03_RNAseq


title: non-inflamed mucosa of CD #3

value: CD #3

value: non-inflamed mucosa


title: HUDEP2_E7_dTAG47_d5

value: immortalized human erythroid progenitor cells

value: nuclear run-on

value: treated with dTAG47 by 5 days


title: HUDEP2_E7_DMSO_d5

value: treated with DMSO by 5 days


title: E8.5_plate1_G02_RNAseq


title: Inflamed mucosa of CD #3

value: Inflamed mucosa


title: plate1_E05_L001_5UDI219-AK4236_RNAseq

value: E7.5 embryo


title: E8.5_plate1_G01_RNAseq


title: HUDEP2_E7_Washout_d4.5

value: treated with dTAG47 by 4 days, and dTAG47 was washed out, PRO-seq was performed 12h after drug wash out.


title: non-inflamed mucosa of CD #2

value: CD #2


title: plate1_E04_L001_AK840-AK4236_RNAseq


title: HUDEP2_E7_dTAG47_d6

value: treated with dTAG47 by 6 days


title: HUDEP2_E7_dTAG47_d4.5

value: treated with dTAG47 by 4.5 days


title: E8.5_plate1_F12_RNAseq


title: Inflamed mucosa of CD #2


title: plate1_E03_L001_5UDI265-AK4236_RNAseq


title: plate1_E02_L001_AK5426-AK4236_RNAseq


title: E8.5_plate1_F11_RNAseq


title: HUDEP2_E7_DMSO_d4.5

value: treated with DMSO by 4.5 days


title: non-inflamed mucosa of CD #1

value: CD #1


title: E8.5_plate1_F09_RNAseq

value: E8.5 embryo


title: B9_DMSO_48h

tag: cell type

tag: rna-source

value: treated with DMSO for 48h


title: B9_dTAG47_48h

value: treated with dTAG47 for 48h


title: E7_dTAG47_48h


title: Inflamed mucosa of CD #1

value: Crohn's disease


title: plate1_D12_L001_AK6249-AK4235_RNAseq


title: E8.5_plate1_F08_RNAseq


title: plate1_E06_L001_AK763-AK4236_RNAseq


title: plate1_E01_L001_AK2831-AK4236_RNAseq


title: E8.5_plate1_F10_RNAseq


title: plate1_D11_L001_AK9170-AK4235_RNAseq


title: E8.5_plate1_F07_RNAseq


title: plate1_D10_L001_AK9404-AK4235_RNAseq


title: plate1_D09_L001_AK6296-AK4235_RNAseq


title: K562, longterm treated with PUH71, replicate 1

city: Düsseldorf

first: Julian

last: Schliehe-Diecks

address: Moorenstr. 5

institute: Uniklinik Düsseldorf

data_processing:
Upload into Galaxy
Adapters were trimmed with FASTQ Trimmer (9 bases of the 5' and 1 base of the 3')
Data quality was assesed via fastqc and aggregated with MultiQC
Reads were aligned to the hg38 reference genome via RNA STAR
Aligned reads were counted with featureCounts and aggregated into a count matrix
edgeR in combination with a gene annotation file was used to normalize the counts (log2 CPM) and to calculate the Fold Change and significance of the genes
Assembly: hg38
Supplementary files format and content: tsv of normalized counts

source_name: blood

growth_protocol: K562 were lentivirally transduced to express CAS9 and a guide RNA, sorted via a fluorescence marker & monoclonally selected

extract_protocol:
RNA was isolated utilizing the Maxwell® RSC simplyRNA Cells Kit (Promega, #AS1390).
Library preparation was carried out following supplier’s guidelines by using the VAHTs Stranded mRNA-Seq Library Prep Kit (Illumina Inc. San Diego, USA). Briefly, total RNA (500 ng) was used for capturing of mRNA, fragmentation, cDNA synthesis, ligation of the adapters and library amplification. Purified libraries were normalized and sequenced on the NextSeq550 (Illumina) with 1x76 bp read setup. Followed by using bcl2fastq2 tool to convert the bcl files to fastq files.

treatment_protocol: K562 were treated with increasing amounts of DMSO / PUH71 to gaine resistant clones. T´hese were monoclonally selected, then again selected and treated

value: blood

value: chronic myeloid leukemia

tag: genotype

value: WT

value: PUH71 longterm


title: plate1_D08_L001_AK7085-AK4235_RNAseq

source_name: Early embryo


title: B9_dTAG47_24h

source_name: HUDEP2 cells

value: immortalized human erythroid progenitor cells

value: nuclear run-on

value: treated with dTAG47 for 24h


title: E8.5_plate1_F05_RNAseq


title: K562, longterm treated with PUH71, replicate 3


title: K562, longterm treated with PUH71, replicate 2


title: K562, longterm treated with DMSO, replicate 3

value: DMSO longterm


title: E8.5_plate1_F06_RNAseq


title: B9_DMSO_24h

value: treated with DMSO for 24h


title: plate1_D07_L001_AK8571-AK4235_RNAseq

value: E7.5 embryo


title: E8.5_plate1_F04_RNAseq


title: E8.5_plate1_F03_RNAseq


title: plate1_D06_L001_AK763-AK4235_RNAseq


title: K562, longterm treated with DMSO, replicate 1


title: E7_dTAG47_24h


title: plate1_D05_L001_5UDI219-AK4235_RNAseq


title: E7_DMSO_48h


title: K562, longterm treated with DMSO, replicate 2


title: E8.5_plate1_F01_RNAseq


title: B9_DMSO_12h

value: treated with DMSO for 12h


title: K562, HSP90 beta KO, replicate 3

value: HSP90 beta KO


title: E8.5_plate1_F02_RNAseq


title: B9_dTAG47_12h

value: treated with dTAG47 for 12h


title: K562, HSP90 beta KO, replicate 1


title: plate1_D04_L001_AK840-AK4235_RNAseq


title: K562, HSP90 beta KO, replicate 2


title: E8.5_plate1_E12_RNAseq

value: E8.5 embryo


title: plate1_D03_L001_5UDI265-AK4235_RNAseq


title: E7_dTAG47_12h


title: E8.5_plate1_E11_RNAseq


title: E7_DMSO_12h


title: K562, HSP90 alpha KO, replicate 3

value: HSP90 alpha KO


title: K562, HSP90 alpha KO, replicate 1


title: E7_DMSO_24h


title: E8.5_plate1_E09_RNAseq


title: B9_DMSO_6h

value: treated with DMSO for 6h


title: plate1_D02_L001_AK5426-AK4235_RNAseq


title: K562, HSP90 alpha KO, replicate 2


title: plate1_C12_L001_AK6249-AK3272_RNAseq


title: E7_dTAG47_6h

value: treated with dTAG47 for 6h


title: plate1_C11_L001_AK9170-AK3272_RNAseq


title: plate1_C10_L001_AK9404-AK3272_RNAseq


title: E8.5_plate1_E07_RNAseq


title: E8.5_plate1_E10_RNAseq


title: B9_dTAG47_3h

value: treated with dTAG47 for 3h


title: E8.5_plate1_E06_RNAseq


title: plate1_C09_L001_AK6296-AK3272_RNAseq


title: E8.5_plate1_E08_RNAseq


title: B9_DMSO_3h

value: treated with DMSO for 3h


title: plate1_D01_L001_AK2831-AK4235_RNAseq


title: E8.5_plate1_E05_RNAseq


title: E7_dTAG47_3h


title: B9_dTAG47_6h


title: E7_DMSO_3h


title: E8.5_plate1_E03_RNAseq


title: plate1_C06_L001_AK763-AK3272_RNAseq


title: plate1_C07_L001_AK8571-AK3272_RNAseq


title: E8.5_plate1_E02_RNAseq


title: E7_0h

value: no treatment


title: plate1_C03_L001_5UDI265-AK3272_RNAseq


title: IgG_2

data_processing:
Raw sequences in FASTQ format were mapped to the spike-in Drosophila genome (dm6) using Bowtie2 (version 2.2.9), with those reads not mapped to the spike genome as input to the primary genome (hg19) using Bowtie2 (version 2.2.9).  Mapped reads were then sorted and indexed using samtools (version 1.9). Duplicate reads, as well as reads mapped to the human blacklisted regions, were filtered out. The resulting reads were then used to generate the bigwig files for visualization in genome browser using the bamCoverage command with the CPM (Count Per Million) normalization method.
Genome_build: hg19
Supplementary_files_format_and_content: Bigwig files were generated for visualization in genome browser.  Values represent read count per million (CPM).

extract_protocol:
About 5-30x10^6 HUDEP-2 cells were cross-linked with fresh 1% formaldehyde (Fisher Scientific) with/without dTAG-47 for 6 min at room temperature (RT) and quenched with 1.25 M glycine for 5 min at RT. Cells were then washed three times in cold phosphate-buffered saline (PBS) containing protease inhibitors and cell pellets flash-frozen in LN2. Nuclei were isolated using the truChIP kit (Covaris Inc.), and chromatin was sheared at 4°C, 140 W, and 200 cycles per burst with 10% duty factor for 8 min (Covaris). Input fraction was saved (1%), and the remaining sheared chromatin was used for ChIP with antibodies specific for an epitope or an immunoglobulin G isotype control (Millipore) in immunoprecipitation buffer [0.1% Triton X-100, 0.1 M tris-HCl (pH 8), 0.5 mM EDTA, and 0.15 M NaCl in 1× Covaris D3 buffer] at 4°C, rotating overnight, followed by incubation with Dynabeads Protein G (10003D, Life Technologies) for 4 hours. The chromatin-bead-antibody complexes were then washed sequentially with three wash buffers of increasing salt concentrations and TE buffer. Chromatin was then eluted using 1% SDS in Tris-EDTA. Cross-linking was reversed by incubation with ribonuclease A (Roche) for 1 hour at 37°C, and with Proteinase K (Roche) at 65oC overnight. DNA was purified with the QIAquick PCR Purification Kit (28104, Qiagen).
ChIPseq libraries were prepared using Swift S2 Acel reagents on a Beckman Coulter Biomek i7 liquid handling platform from approximately 1ng of DNA according to manufacturer’s protocol and 14 cycles of PCR amplification. Finished sequencing libraries were quantified by Qubit fluorometer and Agilent TapeStation 2200.  Library pooling and indexing was evaluated with shallow sequencing on an Illumina MiSeq. Subsequently, libraries were sequenced on an Illumina NovaSeq6000 targeting 40 million 150bp read pairs by the Molecular Biology Core facilities at Dana-Farber Cancer Institute.

tag: chip antibody

value: anti-IgG


title: plate1_C05_L001_5UDI219-AK3272_RNAseq


title: plate1_C04_L001_AK840-AK3272_RNAseq


title: E8.5_plate1_D11_RNAseq


title: E7_DMSO_d4_H3Ac

value: immortalized human erythroid progenitor cells

value: treated with DMSO by 4 days


title: plate1_C02_L001_AK5426-AK3272_RNAseq


title: E8.5_plate1_E04_RNAseq


title: plate1_C08_L001_AK7085-AK3272_RNAseq


title: E8.5_plate1_D10_RNAseq


title: E8.5_plate1_D09_RNAseq


title: IgG_1


title: plate1_B12_L001_AK6249-AK4234_RNAseq


title: E8.5_plate1_E01_RNAseq


title: E8.5_plate1_D12_RNAseq


title: plate1_B10_L001_AK9404-AK4234_RNAseq


title: E7_DMSO_d4_H3K9Ac


title: E8.5_plate1_D06_RNAseq


title: plate1_C01_L001_AK2831-AK3272_RNAseq


title: d0_H3Ac


title: d0_H3K9Ac


title: E7_dTAG47_d4_H3K4me3

value: treated with dTAG47 by 4 days


title: plate1_B08_L001_AK7085-AK4234_RNAseq


title: E8.5_plate1_D03_RNAseq


title: d0_H3K4me3


title: E8.5_plate1_D08_RNAseq


title: plate1_B04_L001_AK840-AK4234_RNAseq


title: plate1_B05_L001_5UDI219-AK4234_RNAseq


title: B9_0h

value: nuclear run-on


title: plate1_B11_L001_AK9170-AK4234_RNAseq


title: E7_dTAG47_d4_H3K9Ac


title: E8.5_plate1_D07_RNAseq


title: plate1_B02_L001_AK5426-AK4234_RNAseq


title: plate1_B01_L001_AK2831-AK4234_RNAseq


title: CMC226 - MP iPSCs

city: Indianapolis

first: Guanglong

last: Jiang

state: IN

department: Medical and Molecular Genetics

institute: Indiana University School of Medicine

data_processing:
Raw reads were mapped by STAR RNA-seq aligner to reference genome hg38
Uniquely mapped reads were assigned to hg38 refGene genes using featureCounts (from subread)
Genome_build: hg38
Supplementary_files_format_and_content: Excel spreadsheet contain raw read counts for each transcript

molecule: polyA RNA

source_name: iPSCs reprogrammed from peripheral blood

growth_protocol:
iPSCs were maintained in mTeSRTM Plus medium on Matrigel-coated plates by a single researcher in order to reduce experimental variability. Medium was replaced daily, and cultures were frequently observed for spontaneous differentiation. Any observed differentiation was removed manually. Each line was passaged one to two times per week using Dispase (1 U/mL in DMEM/F-12; STEMCELL Technologies Inc.) until each target passage number (below) was reached (nine samples total; three cell lines with three target passage numbers each). Low-passage cells were analyzed after 5-10 passages, middle-passage cells after 20-26 passages, and high-passage cells after 30-38 passages. The MycoAlertTM Mycoplasma Detection Kit (Lonza) was utilized to confirm the absence of contamination.
iPSCs from the three different cell lines were induced into sensory neuronal differentiation on Matrigel-coated 6-well plates at ~80% confluence. iPSCs from each cell line were induced at each of three different passage numbers (low, middle, and high), as described above. Differentiation into sensory neurons (iPSC-dSNs) was achieved via a previously published protocol (Cai, Shum, & Chan, 2018). Briefly, differentiation was induced using DMEM/F-12 supplemented with KnockOutTM Serum Replacement (10%), LDN-193189 (0.3 µM), A83-01 (2 μM), CHIR99021 (6 μM), RO4919097 (2 μM), SU5402 (3 μM), and retinoic acid (0.3 μM). Cells were maintained in this induction medium for eight days, with complete medium replacement occurring every other day. On day nine post-induction, cells were dissociated with AccutaseTM (STEMCELL Technologies Inc.). Cells were then strained through a 37 µm mesh and single-cell seeded onto new Matrigel-coated 6-well plates (6 x 105 cells/well) in a maintenance medium consisting of NeurobasalTM Plus (Gibco) medium supplemented with B-27 Plus (1%), neurotrophin-3 (10 ng/mL), brain-derived neurotrophic factor (20 ng/mL), nerve growth factor (20 ng/mL), and glial cell line-derived growth factor (20 ng/mL). This maintenance medium was replaced every other day and cells were allowed to mature for up to an additional 24 days.

extract_protocol: Total RNA was extracted using the QIAGEN RNeasy Mini Kit

value: iPSCs

tag: age post-differentiation (days)


title: plate1_B09_L001_AK6296-AK4234_RNAseq

extract_protocol: Single-cells were sorted  using flow cytometry (E6.5, E7.5) into 96 well PCR plates containing 2.5μl of methylase reaction buffer (1 × M.CviPI Reaction buffer (NEB), 2 U M.CviPI (NEB), 160 μM S-adenosylmethionine (NEB), 1 U μl−1 RNasein (Promega), 0.1% IGEPAL (Sigma)). Samples were incubated for 15 minutes at 37°C to methylate accessible chromatin before the reaction was stopped with the addition of RLT plus buffer (Qiagen) and samples frozen down and stored at -80°C prior to processing. Poly-A RNA was captured on oligo-dT conjugated to magnetic beads and amplified cDNA was prepared according to the G&T-seq and Smartseq2 protocols. The lysate containing gDNA was purified on AMPureXP beads before bisulfite-seq libraries were prepared according to the scBS-seq protocol.

value: E7.5 embryo


title: E7_dTAG47_d4_H3Ac


title: CMC226 - HP iPSC-dSNs

value: iPSC-dSNs


title: E8.5_plate1_D05_RNAseq


title: E8.5_plate1_C09_RNAseq


title: E8.5_plate1_C08_RNAseq


title: E8.5_plate1_D04_RNAseq


title: plate1_A11_L001_AK9170-7UDI268_RNAseq


title: plate1_B07_L001_AK8571-AK4234_RNAseq


title: E7_DMSO_d4_H3K4me3


title: E8.5_plate1_D02_RNAseq


title: plate1_B06_L001_AK763-AK4234_RNAseq


title: E8.5_plate1_D01_RNAseq


title: rsAA-12 - HP iPSCs


title: CMC226 - LP iPSC-dSNs


title: CMC226 - HP iPSCs


title: E8.5_plate1_C12_RNAseq


title: plate1_B03_L001_5UDI265-AK4234_RNAseq


title: CMC226 - MP iPSC-dSNs


title: E8.5_plate1_C11_RNAseq


title: plate1_A08_L001_AK7085-7UDI268_RNAseq


title: E8.5_plate1_C10_RNAseq


title: rsAA-12 - MP iPSC-dSNs


title: E8.5_plate1_C05_RNAseq


title: rsAA-12 - MP iPSCs


title: rsAA-12 - HP iPSC-dSNs


title: CMC226 - LP iPSCs


title: plate1_A12_L001_AK6249-7UDI268_RNAseq


title: plate1_A10_L001_AK9404-7UDI268_RNAseq


title: plate1_A06_L001_AK763-7UDI268_RNAseq


title: H3K36me3-3DA2

city: London

first: LMS

last: Bioinformatics Core

address: Hammersmith Hospital Campus, Du Cane Road

department: Bioinformatics Core

institute: MRC London Institute of Medical Sciences

data_processing:
Reads were cleaned and trimmed using fastq-mcf from the ea-utils suite v1.1.2 to remove adapters, low quality bases and reads, and discard reads shorter than 25 bp after filtering.
Reads were then aligned to the human reference genome (hg19) with bowtie v1.1.1 using best matches parameters (bowtie -v 2 -m 1 --best --strata).
Alignment files were further processed with samtools v1.2 and PicardTools v1.130 to flag PCR and optical duplicates and remove alignments located in Encode blacklisted regions.
Fragment size was estimated in silico for each library using spp v1.10.1.
Genome-wide consistency between replicates was checked using custom R scripts.
Enriched regions were identified for each replicate independently with MACS v2.1.0 with non-IPed genomic DNA as a control (macs2 callpeak --nomodel --shiftsize --shift-control --gsize hs -p 1e-1).
These relaxed peak lists were then processed through the irreproducible discovery rate (IDR) pipeline to generate an optimal and reproducible set of peaks for each histone modification and each time point.
After verifying the consistency between biological replicates, time points and data type, alignements related to biological replicates for a given condition were combined. We then binned the genome in 200bp non-overlapping windows and generated genome-wide signal tracts with deepTools.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig (normalized signal tracks)

source_name: IMR90 ER:RAS

growth_protocol: IMR90 ER:RAS cells were grown in DMEM 10% FBS for 6 days. Media was changed at day 4.

extract_protocol:
10 million cells were crosslinked using 1% formaldehyde for 10 min and libraries were prepared following instructions from manufacturer. H3K36me3 antibody from Cell Signaling Technologies (#4909).
#21024 Accel-NGS 2S Plus DNA library kit (24rxns) plus Adaptor: 26248 2S Set B Indexing Kit.

treatment_protocol: To induce RAS activation, cells were treated with 4OHT [100nM].

tag: days after 4oht

value: IMR90 ER:RAS

value: anti-H3K36me3 (Cell Signaling Technologies, #4909)


title: H3K36me3-4OHT1


title: E8.5_plate1_C06_RNAseq

data_processing:
Libraries were sequenced on the Illumina Nextseq500 platform using the default RTA analysis software.
RNA-seq libraries were aligned to the GRCm38 mouse genome build using HISAT2 (v2.1.0) using options --dta --no-softclipping --no-mixed --no-discordant, yielding a mean of 611,000 aligned reads per cell.
Subsequently, gene expression counts were quantified from the mapped reads using featureCounts with the Ensembl gene annotation (version 87). Only protein-coding genes matching canonical chromosomes were considered. The read counts were log-transformed and size-factor adjusted.
Assembly: GRCm38
Supplementary files format and content: A table of genes (rows) x cells (columns). The first column is the ensembl gene ID

value: E8.5 embryo


title: rsAA-12 - LP iPSC-dSNs


title: rsAA-12 - LP iPSCs


title: E8.5_plate1_C07_RNAseq


title: H3K36me3-3DA1


title: E8.5_plate1_C02_RNAseq


title: plate1_A05_L001_5UDI219-7UDI268_RNAseq


title: plate1_A07_L001_AK8571-7UDI268_RNAseq


title: E8.5_plate1_C04_RNAseq


title: plate1_A04_L001_AK840-7UDI268_RNAseq


title: E8.5_plate1_C03_RNAseq


title: H3K36me3-4OHT2


title: plate1_A02_L001_AK5426-7UDI268_RNAseq


title: E8.5_plate1_B11_RNAseq


title: 06401-2sb - MP iPSCs


title: 06401-2sb - MP iPSC-dSNs


title: plate1_A09_L001_AK6296-7UDI268_RNAseq


title: 06401-2sb - HP iPSC-dSNs


title: E7_DMSO_6h


title: E8.5_plate1_C01_RNAseq


title: H3K36me3-DMSO2

value: DMSO


title: H3K36me3-DMSO1


title: 06401-2sb - HP iPSCs


title: E8.5_plate1_B08_RNAseq


title: TCMK1, TGF-β1 30 min

city: Luzhou

first: Honglian

state: Sichuan

address: No. 182, Chunhui Road, Longmatan District

department: Research Center for Integrative Medicine

institute: the Affiliated Traditional Medicine Hospital of Southwest Medical University

data_processing:
Basecalls were performed using bcl2fastq v2.17 for Novaseq output.
1) Raw fastq sequences were treated with Trimmomatic tools (v 0.36) using the following options: TRAILING: 20, MINLEN: 235 and CROP: 235, to remove trailing sequences below a phred quality score of 20 and to achieve uniform sequence lengths for downstream clustering processes.  2) Sequencing read quality was inspected using the FastQC software. Adapter removal and read trimming were performed using Trimmomatic. Sequencing reads were trimmed from the end (base quality less than Q20) and filtered by length (less than 52).
the clean ChIP-seq reads were mapped to the UCSC mouse genome (GRCm38/mm10) using Bowtie2 (v 2.2.5). Only uniquely mapping reads with at most two mismatches were retained.
bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of DNA fragments at a given genomic coordinate.
Assembly: GRCm38/mm10
Supplementary files format and content: bigwig

growth_protocol: DMEM, 4.5 g/L D-glucose, 10% fetal bovine serum. Cells were cultured at 37C at 5% CO2.

extract_protocol:
Cells were fixed with 1% formaldehyde for 10 min. After quenching and cell lysis, chromatin was prepared with the Magnetic Bead ChIP Kit (Pierce, #26157) following provided instruction.
The library was constructed with the NEBNext® Ultra™DNA Library Prep Kit (NEB,  #E7645S).

treatment_protocol: The cells were starved in 0.5% FBS medium for 6 h followed by stimulation with 5 ng/ml TGF-β1 for 30 min.

value: mouse renal tubular cell


title: E8.5_plate1_B12_RNAseq


title: Input-3DA2

value: none (input)


title: 838_WGBS

data_processing:
Reads were first trimmed using Trim_Galore! (version 0.6.0) with “--paired --clip_R1 10 --clip_R2 10” options
Trimmed reads were then mapped with “bismark” command and deduplicated using Bismark’s “deduplicate_bismark” tool with default parameters
Per-cytosine methylation level was obtained using methylpy (version 1.4.2) “call-methylation-state” tool with “--binom-test True --paired-end True” options
CG methylation levels bigwig files were generated with methlypy (version 1.4.2) with "methylpy allc-to-bigwig --bin-size 10"
genome build: hg38
processed data files format and content: Bigwig, CG methylation levels at 10bp bins

extract_protocol:
DNA bisulfite treatment was performed using the EZ DNA Methylation-Gold Kit (catalog D5005, Zymo Research Corporation) according to the manufacturer's instructions. The recovered bisulfite-converted single-stranded DNA was processed for library construction using the Accel-NGS@Methyl-seq DNA Library kit (catalog 30024, Swift BioSciences) as per manufacturer instructions.
Briefly, using the Adaptase module, truncated adapter sequences were incorporated to the single-stranded DNA in a template-independent reaction through sequential steps. DNA was then enriched using 6 cycles of PCR with primers compatible with Illumina sequencing. The quantity and molecular size of the library was confirmed by Qubit HS DNA assay (ThermoFisher) and Tapestation 2200 system coupled with High Sensitivity D1000 ScreenTapes (Agilent). Illumina 8-nt dual-indices were used for multiplexing. Samples were pooled and sequenced on Illumina NovaSeq S4 sequencer for 150 bp read length in paired-end mode, with an output of 580 million reads per sample.

value: Selected PBMC or BM cells

tag: assay

value: WGBS


title: E8.5_plate1_B06_RNAseq


title: Input-3DA1


title: E8.5_plate1_B10_RNAseq


title: E8.5_plate1_B09_RNAseq


title: NgcKO_Liu_EPSC_noDOX_2

city: New York

first: Jianlong

state: New York

department: Department of Medicine

institute: Columbia University Irving Medical Center

description: Nanog cKO EPSC no Dox

data_processing:
Quality and adaptor trimming of Fastq files were performed using TrimGalore v.0.6.4
Sequence reads were aligned to mm9 (mm9/NCBIM37.67) with STAR v2.7.9a
Read number were counted by htseq v.0.11.2
Differential gene expression was analyzed using DESeq2 v.4.1.1
Assembly: mm9
Supplementary files format and content: Tab-delimited text files including normalized reads counts for each sample.

source_name: mEPSC

growth_protocol: J1 mESCs were cultured on inactive mouse embryonic fibroblast (MEF) feeder cells (approximately 30,000 cells per cm2) or on 0.1% gelatin-coated plates in a serum-based medium with 2i (3µM CHIR99021; 1µM PD0325901) addition, which was prepared as follows: DMEM supplemented with fetal bovine serum (FBS, 15%), recombinant leukemia inhibitory factor (LIF, 1000 units/mL), β-mercaptoethanol (0.1 mM), L-glutamine (2 mM), MEM non-essential amino acids (NEAA, 0.1 mM), nucleoside mix (1%), and Penicillin/Streptomycin (50 U/mL). D-EPSCs were cultured in a base medium of N2B27 prepared as follows: DMEM/F12 and Neurobasal (1:1 ratio), N2 supplement (1X), B27 supplement (1X), Glutamax (1X), β-mercaptoethanol (0.1 mM). The base medium was supplemented with KSR (5%), NEAA (0.1 mM), LiF (1000 units/mL), CHIR99021 (3µM); dimethindene maleate (DiM, 2µM); and minocycline hydrochloride (MiH, 2µM). L-EPSCs were cultured on feeder cells or gelatin-coated plates in a base medium prepared as follows: DMEM/F12, KnockOut Serum Replacement (KSR, 20%), L-glutamine (2 mM), NEAA (0.1 mM), β-mercaptoethanol (0.1 mM). The base medium was supplemented with Lif (1000 units/mL), CHIR99021 (3µM), PD0325901 (1µM), A-419259 (0.3µM), XAV939 (5µM), JNK inhibitor VIII (4µM), SB203580 (10µM).

extract_protocol:
Total RNA from ESCs and EPSCs replicates were extracted using Trizol according to the manufacturer’s protocol.
RNA-seq libraries were prepared manually using Universal Plus mRNA-Seq with NuQuant kit, according to the manufacturer’s protocol.

treatment_protocol: Doxycycline was added to cells to deplete expression of Sox2, Oct4, or Nanog.

value: Nanog Tet-off knockin

value: mEPSC


title: plate1_A01_L001_AK2831-7UDI268_RNAseq

city: Cambridge

first: Felix

institute: Altos Labs


title: 798_WGBS


title: Input-4OHT2


title: 06401-2sb - LP iPSC-dSNs


title: NgcKO_Liu_EPSC_noDOX_1

description: Nanog cKO EPSC no Dox


title: Input-4OHT1


title: shAHCY4_63

data_processing:
Illumina Casava version 1.8.2 was used for basecalling.
RNA-seq reads were aligned to human genome hg19 (GRCh37) using TopHat2 version 2.0.11 with the parameter "--library-type=fr-unstranded"
Gene annotation information from Ensembl GRCh37 were employed
Rsubread featureCounts version 1.18.0 was used to generate gene-based read counts matrix
Genome_build: hg19
Supplementary_files_format_and_content: CSV file containing gene-based read counts matrix

extract_protocol:
Total RNA was extracted using Trizol and the Rneasy isolation kit (Qiagen).
Smart-Seq2 protocol using Nextera XT Dual Indexing Kit.

tag: passage

value: shAHCY_4 (+4OHT)


title: plate1_A03_L001_5UDI265-7UDI268_RNAseq


title: NgcKO_Liu_EPSC_DOX_2

description: Nanog cKO EPSC Dox

value: Doxycycline


title: E8.5_plate1_B03_RNAseq


title: 06401-2sb - LP iPSCs


title: input-DMSO2


title: shAHCY4_62


title: 496_WGBS


title: 933_WGBS


title: E8.5_plate1_B07_RNAseq


title: NgcKO_Liu_EPSC_DOX_1

description: Nanog cKO EPSC Dox


title: input-DMSO1


title: shAHCY4_64


title: shAHCY2_60

value: shAHCY_2 (+4OHT)


title: E8.5_plate1_B01_RNAseq


title: shAHCY2_59


title: 504_WGBS


title: E8.5_plate1_B04_RNAseq


title: ZHBTc4_Liu_EPSC_noDOX_2

description: Oct4 cKO EPSC no Dox

value: Oct4 Tet-off knockin


title: NgcKO_ESC_2

description: Nanog cKO ESC

source_name: mESC

value: mESC


title: NgcKO_ESC_1

description: Nanog cKO ESC


title: 424_WGBS


title: E8.5_plate1_A11_RNAseq


title: shAHCY4_61


title: HSPC-822_Hi-C

data_processing:
Paired-end reads were first trimmed by Trim_Galore! (version 0.6.0) to remove adapters and low-quality bases with parameter “--paired”
Trimmed reads were then mapped to human genome GRCh38 by BWA MEM (version 0.7.17-r1198) with parameter “-SP5M”
Mapped reads were deduplicated with “pairtools dedup” (version 0.3.0)
Reads mapped to the same MboI restriction fragment or in outward direction are removed for downstream analysis.
Final pairs files were generated using Pairtools (version 0.3.0)
Genome_build: hg38
Supplementary_files_format_and_content: Pairs format, chromatin interaction intensity for pairs of genomic loci

growth_protocol: HL60 (ATCC CCL-240) and Kasumi-1 (ATCC CRL-2724) cells were given to us as a gift from Dr Sinisa Dovat lab, which are new vials purchased from ATCC. Cells are cultured following the manufacturer’s culture method. Kasumi is cultured with RPMI-1640 (Gibco, 11875093) with 20% FBS. HL60 is cultured with Iscove's Modified Dulbecco's Medium (IMDM) (ATCC® 30-2005).

extract_protocol:
One to two million cryopreserved primary samples or cell lines in cell culture were spinned down with 500g and resuspended in 1ml/million RPMI 1640 medium with 10% fetal bovine serum, immediately crosslinked with 37% formaldehyde (MilliporeSigma 252549) to a final concentration of 2%, and incubated at room temperature for 10 minutes on a tube revolver at 16 rpm to mix, quenched by 2.5M glycine solution with a final concentration of 0.2M and incubated in room temperature for 5 minutes on revolver. Cells were pelleted by centrifuge at 500g at 4°C for five minutes and washed once with 1ml cold 1X PBS by centrifuge at 500g at 4°C for five minutes and the supernatant was discarded. Cells were lysed to extract nuclei with 250ul lysis buffer (10mM Tris-HCl pH8.0, 10mM NaCl, 0.2% Igepal CA630) mixed with 50ul 50x protease inhibitor (Sigma, P8340) and incubated on ice for 15 minutes, centrifuge to pellet at 2500g, 4°C for five minutes and washed with 500ul lysis buffer. Cell pellets were resuspended in 50ul 0.5% sodium dodecyl sulfate (SDS) and incubated at 62°C for 10 minutes, quenched by 145ul water and 25ul 10% Triton X-100 (Sigma, 93443), and incubated at 37°C for 15 minutes. 25μl of 10X NEBuffer2 (NEB B7207) and 100 unit of MboI restriction enzyme (NEB, R0147) was then added to the reaction for overnight DNA digestion at 37°C on the tube revolver. The digestion was then quenched by incubation at 62°C for 20 minutes. DNA was then end repaired and Biotin labeled with 50ul fill-in master mix (37.5μl of 0.4mM biotin-14-dATP (Life Technologies, 19524-016), 1.5μl of 10mM dCTP, 1.5μl of 10mM dGTP, 1.5μl of 10mM dTTP, 8μl of 5U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210) and incubated at 37°C for 1.5 hours.Then DNA was ligated with 900ul of ligration master mix (669ul water, 120μl of 10X NEB T4 DNA ligase buffer (NEB, B0202), 100μl of 10% Triton X-100, 6μl of 20mg/ml Bovine Serum Albumin (MilliporeSigma B8667), 5μl of 400 U/ μl T4 DNA Ligase (NEB, M0202) ), incubated at room temperature for 4 hours with slow rotation. Decrosslink DNA by adding 50ul of 20mg/ml proteinase K (QIAGEN 19133) and 120ul of 10% SDS, incubated at 55°C for 30 minutes. Quench the reaction by adding 130μl of 5M sodium chloride and incubate at 68°C overnight. Precipitate DNA by adding 1.6X volume of pure ethanol and 0.1X volume of 3M sodium acetate, pH 5.2 (MilliporeSigma S7899), incubate at  -80°C for at least half hour, and centrifuge at max speed, 2°C for 15 minutes to discard supernatant. Wash DNA once with 700ul 80% ethanol. Dissolve dried DNA pellet in 130ul 10 mM Tris-HCl, pH 8. Sonicate the solution to shear DNA to average size of 300-500bp with Covaris sonicator, with parameters set as followings: PIP 140, duty factor 10, burst 200, and duration 58s-80s. Run 4ul sheared DNA in 16ul water on a 2% agarose to verify the size.
Pull down biotin-labeled DNA by washing 150μl of 10mg/ml Dynabeads MyOne Streptavidin T1 beads (Life technologies, 65602) with 400μl of 1X Tween Washing Buffer (TWB: 5mM Tris-HCl (pH 7.5); 0.5mM EDTA; 1M NaCl; 0.05% Tween 20), discard the solution. Resuspend the beads in 300μl of 2X Binding Buffer (10mM Tris-HCl (pH 7.5); 1mM EDTA; 2M NaCl) and add to the sheared DNA. Incubate at room temperature for 15 minutes with rotation. Separate beads and discard the supernatant with a magnetic rack. Wash beads with 600ul TWB buffer twice. End repair of sheared DNA by resuspending beads in 100ul 1X NEB T4 DNA ligase buffer (NEB, B0202), separating the beads, and resuspending in end repair master mix (88μl of 1X NEB T4 DNA ligase buffer with 10mM ATP (NEB B0202S), 2μl of 25mM dNTP mix, 5μl of 10U/μl NEB T4 PNK (NEB, M0201), 4μl of 3U/μl NEB T4 DNA polymerase I (NEB, M0203) , 1μl of 5U/μl NEB DNA polymerase I, Large (Klenow) Fragment (NEB, M0210) ), and incubation at room temperature for half hour. Beads were washed twice with 500ul TWB buffer and resuspended in 100μl 1X Quick ligation reaction buffer (NEB, B6058), recollected, and proceeded with dATP attachment by resuspended in 100μl master mix (90μl of 1X NEBuffer 2, 5μl of 10mM dATP, 5μl of 5U/μl NEB Klenow exo minus (NEB, M0212)), incubated at 37°C for 30 minutes. Beads were washed twice with 500ul TWB buffer and resuspended in 100μl 1X Quick ligation reaction buffer (NEB, B6058), recollected, proceeded with adaptor ligation through resuspension in  50μl of 1X NEB Quick ligation reaction buffer, 2μl of NEB DNA Quick ligase (NEB, M2200), and 3ul of Illumina adaptor of choice, incubated in room temperature for 15 minutes. Beads were washed by 600ul TWB buffer and 100ul 1X Tris buffer, resuspended in 50ul 1X Tris buffer, heated on 98°C for 10 minutes to elute the DNA off the beads. Beads were discarded. Size selection was performed to remove small DNA fragments by adding 0.8X-0.9X KAPA beads to the DNA elution, incubation at room temperature for 5 minutes, and beads were collected with supernatant discarded. Wash beads twice with 500ul 80% ethanol and elute beads in 50ul 1X Tris buffer. Library amplification was performed with 4-12 cycles of PCR with KAPA 2X library mix. Size selection was performed to remove small and large fragments using KAPA beads and maintain DNA fragments of 150bp-500bp.  Libraries were sequenced as 150 bp paired-end reads with a raw sequencing depth between 300 million to 700 million read pairs per sample on platform Novaseq.

treatment_protocol: 5-azacytidine (MilliporeSigma A2385-100MG) was dissolved in DMSO to make 100mM stock solution, aliquoted and stored in -80°C. Working solutions (0.5uM-10uM) was made from further dilution of stock solution using complete cell culture media. Media was changed every 24 hours with freshly made 5-AZA solution. Dead cells are removed by Ficoll-Paque PLUS density gradient media at the end of cell culture before cells are further processed for any profiling experiments.

value: Hi-C


title: E8.5_plate1_B05_RNAseq


title: HSPC-341_Hi-C


title: ZHBTc4_Liu_EPSC_DOX_2

description: Oct4 cKO EPSC Dox


title: E8.5_plate1_B02_RNAseq


title: ZHBTc4_ESC_2

description: Oct4 cKO ESC


title: ZHBTc4_Liu_EPSC_DOX_1

description: Oct4 cKO EPSC Dox


title: E8.5_plate1_A08_RNAseq


title: shAHCY2_58


title: E8.5_plate1_A12_RNAseq


title: EV+_55

value: senescent cells (EV+4OHT)


title: 1071_WGBS


title: ZHBTc4_Liu_EPSC_noDOX_1

description: Oct4 cKO EPSC no Dox


title: shAHCY2_57

value: IMR90 ER:RAS


title: EV+_56


title: E8.5_plate1_A10_RNAseq


title: E8.5_plate1_A09_RNAseq


title: EV+_54


title: HSPC-213_Hi-C


title: 1324_WGBS


title: 2TS22C_Liu_EPSC_DOX_2

description: Sox2 cKO EPSC Dox

value: Sox2 Tet-off knockin


title: 933_Hi-C


title: EV_52

value: non-senescent cells (EV)


title: E8.5_plate1_A06_RNAseq

value: E8.5 embryo


title: 2TS22C_Liu_EPSC_DOX_1

description: Sox2 cKO EPSC Dox


title: E8.5_plate1_A07_RNAseq


title: 2TS22C_Liu_EPSC_noDOX_2

description: Sox2 cKO EPSC no Dox


title: ZHBTc4_ESC_1

description: Oct4 cKO ESC


title: HSPC-050_Hi-C


title: 798_Hi-C


title: EV+_53


title: EV_51


title: 838_Hi-C


title: 496_Hi-C


title: E8.5_plate1_A04_RNAseq


title: EV_50


title: 2TS22C_ESC_1

description: Sox2 cKO ESC


title: E8.5_plate1_A03_RNAseq


title: EV_49


title: E8.5_plate1_A05_RNAseq


title: RNA_J1-Deng-EPS-2

description: D-EPSC

value: WT


title: 424_Hi-C


title: E8.5_plate1_A02_RNAseq


title: 3DA_32

value: 3DA + 4OHT


title: 1324_Hi-C

value: Selected PBMC or BM cells


title: RNA_J1-Deng-EPS-1

description: D-EPSC


title: 504_Hi-C


title: E7.5_tet_chimera_plate3_H12_RNAseq

value: E7.5 embryo


title: 1071_Hi-C


title: RNA_J1-Liu-EPS-2

description: L-EPSC


title: RNA_J1-Liu-EPS-1

description: L-EPSC


title: 3DA_30


title: E7.5_tet_chimera_plate3_H11_RNAseq


title: 3DA_29


title: E7.5_tet_chimera_plate3_H10_RNAseq