title: 4Cseq Dlx5/6UCE bait Sox2flfl Dlx5/6cre pos

city: Chicago

first: Jhumku

last: Kohtz

state: Illinois

address: 303 E Chicago

department: Neurology

institute: Northwestern University

data_processing:
Library strategy: 4C-Seq
Cut&Run data processing
Paired-end Cut&Run reads of different transcription factors and their respective Ig-controls from Evf2+/+ and Evf2TS/TS Dlx5/6UCE samples were first mapped on mm9 genome using Bowtie2 v2.1.0 (Langmead et al. 2012) with options'--local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700'. We used the Picard toolkit command 'MarkDuplicates' to mark PCR duplicates and remove them from the final mm9 genome mapped bam files. Next, we separated the sequence fragments into ≤ 120 and ≥ 150-bp size classes that provided the mapping of the local vicinity of a DNA-binding protein. The base-pair sizes can vary depending on the steric access to the DNA by the tethered MNase (Skene et al.). Fragments mapping to repeat elements were removed, and replicates were joined before peak calling. The peak calling was performed using MACS2 (Zhang et al.) callpeak options'-t -c –f BED -g mm --keep-dup all --bdg –nomodel --slocal 500 --llocal 5000 –-extsize 120/150'. An FDR cutoff of 0.05 was used to call the final set of peaks (Janssens et al.). Differential Cut&Run analysis of transcription factor binding peaks between two Dlx5/6UCE conditions was performed using MACS2 program by treating one of the samples as the "control" for the other.
Sox2flfl +/cre 4C-seq differential data analysis
4C reads were first mapped at the EcoRI restriction enzyme cut sites on chromosome 6 of mm9 reference genome using Bowtie2 v2.1.0 (Langmead and Salzberg 2012). 4C contact counts were generated from the Bowtie2 mapped bam files using FourCSeq program (Klein et al. 2015). We then performed a DESeq2 (Love et al. 2014) based differential contact count analysis on these sites to identify Sox2flfl sites (p-adjusted value < 0.05 and a log2 fold change ≥ 2 for positively regulated (cre) or ≤ -2 for negatively regulated (+) 4C interaction sites with non-zero counts in at least two of the replicates in a differential condition. We also performed the 4Cseq analysis using the FourCSeq program. FourCSeq program models the overall decreasing interaction frequency with genomic distance by fitting a smooth monotonically decreasing function to suitably transformed count data. With this transformed and normalized count data, FourCSeq performs differential analysis between conditions to get significant differential interactions. We applied FourCSeq on our Sox2flfl + and Sox2flfl cre 4Cseq samples and retrieved Sox2 regulated 4C interactions (p-adjusted value < 0.05 and an absolute log2 fold change ≥ 2). To avoid method-specific biases, we retained a common set of 244 4C differential sites.
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph files contains logfold enrichment, qvalue signal tracks from Cut&Run samples and 4C count and differential peaks

source_name: Sox2flfl cre pos E13.5GE

extract_protocol:
4C was performed as previously described (van de Werken et al. 2012), with some modifications. GEs from Sox2 fl/fl Cre+ and Sox2 fl/fl Cre- single embryos were dissected in L15 and kept in separate tubes. A single cell suspension was obtained through gentle pipetting of the tissue in 250 μl of L15. Cells (approximately 2 x106 cells per embryo) were transferred to a tube containing 5 ml 2% paraformaldehyde/10% Fetal Bovine Serum (FBS) and incubated with rotation for 10 min at room temperature. 710 μl of 1M glycine were added to quench the formaldehyde and tubes were placed on ice. Cells were pelleted by centrifugation at 1300 rpm for 8 min at 4°C. The supernatant was removed and cells were gently resuspended in 2.5 ml ice-cold 4°C Lysis Buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% Triton X-100, protease inhibitors) and incubated for 10 min on ice.  Cells were pelleted by centrifugation at 1800 rpm for 5 min at 4°C. The supernatant was removed, and the cells were washed with 1 ml ice-cold 1x PBS by gentle resuspension. Cells were transferred to 1.5 ml microcentrifuge tubes and centrifuged at 2,400 rpm for 2 min at 4°C. The supernatant was removed and the cell pellets were flash frozen in liquid nitrogen and stored at -80°C.   The cell pellets were resuspended in 440 μl of molecular grade water and 60 μl of Cutsmart Buffer (New England Biolabs) were added. Tubes were incubated at 37°C and 15 μl of 10% SDS were added. Samples were incubated for 1 hour at 37°C while shaking at 900 rpm. 75 ul of 20% Triton X-100 were added to the samples and incubated at 1 hour at 37°C while shaking at 900 rpm. 200 U of EcoRI-HF were added to the samples and incubated overnight at 37°C while shaking. The next day, 200 U of EcoRI-HF were added and incubated overnight at 37°C with shaking. Complete digestion was confirmed by agarose gel electrophoresis. If undigested DNA was still present, another 200 U of restriction enzyme was added and incubated overnight. The enzyme was inactivated at 65°C for 20 min. Samples were transferred to a 15 ml conical tube. 100 U of T4 DNA Ligase were added (2 ml reaction volume) and incubated overnight at 16°C. The next day, 500 μl of molecular grade water, 50 μl fresh T4 ligase buffer and 100 U of T4 DNA ligase were added to the samples and incubated overnight at 16°C. Complete ligation was confirmed by agarose gel electrophoresis. The DNA was extracted using standard ethanol precipitation procedures (van de Werken et al. 2012).    The DNA pellet was resuspended in 450 μl of molecular grade water, then 50 μl of DpnII buffer and 50 U of DpnII were added and incubated at 37°C overnight. Complete digestion was confirmed by agarose gel electrophoresis. The enzyme was inactivated at 65°C for 20 min. Samples were transferred to a 50 ml conical tube and ligation was performed using 100 U of T4 DNA ligase in a total volume of 3.5 ml at 16°C overnight. The DNA was precipitated using standard ethanol precipitation procedures. The samples were purified using the Qiaquick PCR purification kit (2 columns per sample).
The following steps were performed to generate the 4C library for sequencing. First, overhangs were added to the 4C template using PCR amplification with primers containing the bait sequence, as follows, PCR reaction: 200 ng 4C template, 0.2 mM dNTPs, 35 pmol Primer Dlx5/6UCE-Fwd, 35 pmol Primer Dlx5/6UCE-Rev, 1.75 U Expand Long Template Enzyme Mix (Roche), 1X Buffer I. PCR cycles: 94°C - 2 min, 94°C – 10 sec, 55°C - 1min, 68°C - 3 min, 29 cycles, 68°C - 5min. The PCR product was purified using the High Pure PCR Product Purification Kit (Roche). Then, the 4C DNA containing the overhangs was used as template for a second PCR that adds index sequences and Illumina sequencing adapters to generate the 4C library for sequencing. PCR reaction (50 μl): 225 ng DNA template, 0.5 mM dNTPs, 5μl Nextera XT Index1 primer (N7XX, Illumina), 5 μl Nextera Index 2 primer (S5XX, Illumina), 3.5 U Expand Long Template Enzyme Mix (Roche), 1X Buffer I. PCR cycles: 94°C - 5 min, 94°C – 10 sec, 55°C – 30 sec, 68°C - 1 min, 8 cycles, 68°C - 7min.The PCR product was purified using the High Pure PCR Product Purification Kit (Roche).
mouse E13.5 ganglionic eminences were dissected, and single cell suspensions were processed for CUT&RUN, mouse E13.5 ganglionic eminences were dissected, and  processed for 4Cseq

value: E13.5 ganglionic eminence

value: not specified


title: 4Cseq Dlx5/6UCE bait Sox2flfl cre neg

status: Public on Feb 23 2021

type: SRA

country: USA

library_source: genomic

molecule: genomic DNA

organism: Mus musculus

source_name: Sox2flfl cre neg E13.5GE

tag: cell type

tag: strain


title: 200_ng_U023_SR

city: South San Francisco

first: Rajat

last: Bhatnagar

state: CA

address: Two Tower Place, Suite 940

institute: Verge Genomics

description: pool of RNA from 10 different human cancer cell lines

library_source: transcriptomic

data_processing:
Reads were aligned with STAR version 2.7.2  to the reference genome GRCh38 with Ensembl gene annotations. We required genes to have a minimum 3 counts per million in at least 20% of samples. Genes not satisfying this requirement were removed.  After filtering, approximately 12,000 genes remained.  To compare absolute gene expression, reads were normalized via the regularized log transform.
Genome_build: GRCh38

molecule: total RNA

organism: Homo sapiens

source_name: Universal Human Reference RNA (UHRR)

extract_protocol:
1. Swift RNA Library Prep. 100, 50 and 10 ng of UHRR was used as direct input for NEBNext Ploy(A) mRNA Magnetic Isolation Module (New England Biolabs, E7490). In the final elution step, mRNA was eluted off the beads into the Swift RNA Library Kit Fragmentation Buffer. Libraries were prepared and indexed (Swift Unique Dual Indexing Kit, X9096) using Swift recommended protocol. cDNA libraries were diluted 1:5 in low TE buffer and analyzed on Agilent 4200 TapeStation using D5000 HS DNA screentape. Libraries that gave yields < 4 nM were amplified using the Swift Library Amplification Primer Kit (88196).
2. Swift Rapid RNA Library Prep. 200, 100 and 50 ng of UHRR was used as direct input for NEBNext Ploy(A) mRNA Magnetic Isolation Module (New England Biolabs, E7490). In the final elution step, mRNA was eluted off the beads into the Swift RNA Library Kit Fragmentation Buffer. Libraries were prepared and indexed (Swift Unique Dual Indexing Kit, X9096) using Swift recommended protocol. cDNA libraries were diluted 1:1 in low TE buffer and analyzed on Agilent 4200 TapeStation using D5000 DNA screentape.
3. Illumina TruSeq Stranded mRNA Library Prep. 500, 200, 100 and 50 ng of UHRR was used as direct input for the Illumina TruSeq Stranded mRNA (20020594). Libraries were prepared and indexed (IDT for Illumina-TruSeq RNA UD Indexes, 20022371) as outlined in the TruSeq Stranded mRNA Reference Guide.

value: Adenocarcinoma (mammary gland), Melanoma, Hepatoblastoma (liver), Liposarcoma, Adenocarcinoma (cervix), Histiocytic lymphoma (macrophage; histocyte), Embryonal carcinoma (testis), Lymphoblastic leukemia(T lymphoblast), Glioblastoma (brain), Plasmacytoma/myeloma (B lymphocyte)

tag: commercial source

value: ThermoFisher (QS0639)

tag: method

value: Swift Rapid library prep


title: CTCF_4h_ChIPseq_Rep1

city: Philadelphia

first: Jing

last: Luan

address: 3615 Civic Center Blvd, ARC 315

institute: University of Pennsylvania

data_processing:
Bowtie 1.1.0 was used to align sequences (Langmead and Salzberg, 2012) to the mm9 reference genome. Reads with more than one mismatch or multiple alignments were excluded.
Significantly enriched regions were called using MACS2 version 2.1.0 (Y. Zhang et al., 2008) with the following parameters: P = 10−5, extsize = 300 and local lambda = 100,000 using whole-cell extract input controls.
Reads for the bigwigs were RPM normalized.
Genome_build: mm9

source_name: CTCF-AID-mCherry+TIR

growth_protocol: G1E-ER4 (Weiss et al., 1997) derived single-cell clones were grown in IMDM+15% FBS, penicillin/streptomycin, kit ligand, monothioglycerol and Epoetin alpha in a standard tissue culture incubator at 37 degrees with 5% CO2 (as described in Weiss et al., 1997).

extract_protocol:
Lysates were prepared from sonicated nuclei and histone-DNA complexes were isolated with indicated antibodies.
ChIP-seq libraries were prepared using Illumina’s TruSeq ChIP sample preparation kit (Illumina, catalog no. IP-202-1012) according to manufacturer’s specifications, with the addition of size selection (left side at 0.9x, right side at 0.6x) using SPRIselect beads (Beckman Coulter, catalog no. B23318). Library size was determined (average 351 bp, range 333-372 bp) using the Agilent Bioanalyzer 2100, followed by quantitation using real-time PCR using the KAPA Library Quant Kit for Illumina (KAPA Biosystems catalog no. KK4835). Libraries were then pooled and sequenced (1x75bp) on the Illumina NextSeq 500 platform according to manufacturer’s instructions. Bclfastq2 v 2.15.04 (default parameters) was used to convert reads to fastq.

treatment_protocol: Cells were treated with either auxin (1mM), Triptolide (1uMx5h) or DRB (75uMx7h). See Description column for Samples.

value: ES-derived erythroid progenitor cells

tag: chip antibody

value: Millipore 07-729

tag: treatment

value: 4h auxin


title: PIT_P5-PIT_P6-B3-AD007

city: La Jolla

first: Joseph

middle: R

last: Ecker

address: 10010 North Torrey Pines Road

department: Genomic Analysis Laboratory

institute: HHMI-Salk-Institute

data_processing:
Library strategy: snmC-seq2
Reads trimed with Cutadapt for adapters and low quality bases (Phred score >= 20)
The resulting files are mapped to in-silico bisulfite converted mm10 reference genome using Bismark.
The raw BAM file will be filtered using samtools to filter reads by MAPQ > 10 and use Picard to remove PCR duplicates
Tab-delimited files (ALLC files) describing the quantity of unmethylated and methylated cytosine base calls will be generated with in-house developing package using the filtered BAM files
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited files (ALLC files) describing the quantity of unmethylated and methylated cytosine base calls. Columns are: 1. Chromosome, 2. Position, 3. Strand, 4. Cytosin Context, 5. methylated base call, 6. total mapped base call, 7. not used, place holder column

source_name: C57BL/6J Male P56 Mouse Brain

growth_protocol: All mice are C57BL/6 Male, brain dissected in P56

extract_protocol:
The snmC-seq2 protocol was applied to individual nuclei isolated from pituitary pooled from 30 adult male C57BL/6 mice
Library prepared following protocol discribed in Luo, Chongyuan, Angeline Rivkin, Jingtian Zhou, Justin P. Sandoval, Laurie Kurihara, Jacinta Lucero, Rosa Castanon, et al. 2018. “Robust Single-Cell DNA Methylome Profiling with snmC-seq2.” Nature Communications 9 (1): 3824.

tag: tissue

value: Pituitary

tag: age

tag: Sex

value: male


title: PROseq_4h_R3

description:
4h auxin; replicate #3
2008

data_processing:
Seqtk (https://github.com/lh3/seqtk/blob/master/README.md) was run to trim the 6-base UMI.
Reads were mapped to both fly (dm6) and mouse (mm9) genomes, with only those mapped exclusively to one genome kept for downstream analysis.
Only primary alignments with mapq score at least 20 and without INDEL were reported and saved in bam files.
Duplicated reads with the same UMI and aligned to the same genomic loci were removed.
Bigwig files were generated and normalized using the sample size factor calculated by DEseq2.
Genome_build: mm9

extract_protocol:
Nuclei were isolated for PRO-seq, followed by run-on reactions, nascent RNA isolation, and cDNA library preparaion as described in the paper: Mahat, D.B., Kwak, H., Booth, G.T., Jonkers, I.H., Danko, C.G., Patel, R.K., Waters, C.T., Munson, K., Core, L.J., Lis, J.T., 2016a. Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq). Nat Protoc 11, 1455–1476. doi:10.1038/nprot.2016.086
library srategy: PRO-Seq

treatment_protocol: Auxin concentraton was 1mM. See Description column for Samples.


title: PROseq_4h_R2

description:
4h auxin; replicate #2
1699


title: PIT_P1-PIT_P2-F5-AD006


title: 200_ng_U022_SR


title: PROseq_0h_R3

description:
No treatment; replicate #3
2007

value: No treatment


title: 12 CUT&RUN IgG

data_processing:
Library strategy: Cut&Run
Cut&Run data processing
Paired-end Cut&Run reads of different transcription factors and their respective Ig-controls from Evf2+/+ and Evf2TS/TS Dlx5/6UCE samples were first mapped on mm9 genome using Bowtie2 v2.1.0 (Langmead et al. 2012) with options'--local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700'. We used the Picard toolkit command 'MarkDuplicates' to mark PCR duplicates and remove them from the final mm9 genome mapped bam files. Next, we separated the sequence fragments into ≤ 120 and ≥ 150-bp size classes that provided the mapping of the local vicinity of a DNA-binding protein. The base-pair sizes can vary depending on the steric access to the DNA by the tethered MNase (Skene et al.). Fragments mapping to repeat elements were removed, and replicates were joined before peak calling. The peak calling was performed using MACS2 (Zhang et al.) callpeak options'-t -c –f BED -g mm --keep-dup all --bdg –nomodel --slocal 500 --llocal 5000 –-extsize 120/150'. An FDR cutoff of 0.05 was used to call the final set of peaks (Janssens et al.). Differential Cut&Run analysis of transcription factor binding peaks between two Dlx5/6UCE conditions was performed using MACS2 program by treating one of the samples as the "control" for the other.
Sox2flfl +/cre 4C-seq differential data analysis
4C reads were first mapped at the EcoRI restriction enzyme cut sites on chromosome 6 of mm9 reference genome using Bowtie2 v2.1.0 (Langmead and Salzberg 2012). 4C contact counts were generated from the Bowtie2 mapped bam files using FourCSeq program (Klein et al. 2015). We then performed a DESeq2 (Love et al. 2014) based differential contact count analysis on these sites to identify Sox2flfl sites (p-adjusted value < 0.05 and a log2 fold change ≥ 2 for positively regulated (cre) or ≤ -2 for negatively regulated (+) 4C interaction sites with non-zero counts in at least two of the replicates in a differential condition. We also performed the 4Cseq analysis using the FourCSeq program. FourCSeq program models the overall decreasing interaction frequency with genomic distance by fitting a smooth monotonically decreasing function to suitably transformed count data. With this transformed and normalized count data, FourCSeq performs differential analysis between conditions to get significant differential interactions. We applied FourCSeq on our Sox2flfl + and Sox2flfl cre 4Cseq samples and retrieved Sox2 regulated 4C interactions (p-adjusted value < 0.05 and an absolute log2 fold change ≥ 2). To avoid method-specific biases, we retained a common set of 244 4C differential sites.
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph files contains logfold enrichment, qvalue signal tracks from Cut&Run samples and 4C count and differential peaks

source_name: Evf1TSTS E13.5GE

extract_protocol:
Cut&Run was performed as previously described (Skene et al. 2018) with some modifications. The number of samples was determined according to the number of antibodies to be tested and the number of replicates (2 replicates per antibody and 2 replicates for IgG). The appropriate volume of Concanavalin A magnetic beads (10 μl per sample) were mixed into 1.5 ml of ice-cold Binding Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1mM MnCl2) and placed on a magnetic stand for 2 min. The supernatant was removed, and the beads were washed with 1.5 ml Binding Buffer. After magnetic separation of the beads, the supernatant was removed, and a volume of Binding Buffer equal to the initial beads volume (10 μl per sample) was added to the beads. The beads were placed on ice.    E13.5 ganglionic eminences were isolated from embryos in L15 medium (6 Embryos per genotype). Tissues were pooled for each genotype, triturated by pipetting, and filtered through a cell-strainer capped 5 ml polystyrene round- bottom tube (BD Falcon) to generate single-cell suspensions. Cells were counted using the Luna Automated Cell Counter (Logos Biosystems). The appropriate volume of cells to obtain 250,000 cells per sample was centrifuged at 600 g for 3 min at 4°C. The supernatant was removed and the cell pellet was gently resuspended in ice-cold Wash Buffer (20mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, EDTA-free protease inhibitor cocktail). The cells were centrifuged at 600 g for 3 min at 4°C. The supernatant was removed and cells were gently resuspended in 1 ml of Wash Buffer. The Concanavalin A bead suspension was added to the cells, while gently vortexing (~1000 rpm), and the tube was incubated with rotation for 10 min at 4°C. The cells/beads suspension was split into aliquots, according to the number of samples previously determined. Tubes were placed on a magnetic stand and the supernatant removed. 50 ul of Antibody Buffer (20mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 2 mM EDTA, 0.02% digitonin, EDTA-free protease inhibitor cocktail) containing 0.5 μg of antibody was added to each tube, while gently vortexing. The samples were incubated with rotation for 2 hours at 4°C. The samples were centrifuged for 5 seconds at 1000 rpm, and placed on the magnetic stand. The supernatant was removed, and the pellet resuspended gently in 1 ml ice-cold Digitonin Buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.02% digitonin, EDTA-free protease inhibitor cocktail). The Digitonin Buffer wash was repeated once. Tubes were placed on the magnetic stand, the supernatant removed and 50 μl of pA-Mnase solution (final concentration 700 ng/ml in Digitonin Buffer) were added to each tube while gently vortexing. The samples were incubated with rotation for 1 hours at 4°C. The samples were centrifuged for 5 sec at 1000 rpm and placed on the magnetic stand. The supernatant was removed and the pellets were washed in ice-cold Digitonin Buffer two times, as described above. After removal of supernatant from washes, 150 μl of Digitonin Buffer were added to each sample while gently vortexing. Tubes were placed on a metal block on ice (0°C) for 5 min. Tubes were removed from ice briefly to add 3 μl 100 mM CaCl2 while gently vortexing, then tubes were returned to 0°C for 30 min. 100 ul of Stop Buffer (340 mM NaCl, 20 mM EDTA, 4mM EGTA, 0.02 % digitonin, 0.05 mg/ml RNAseA, 0.05 mg/m, 2 pg/ml heterologous spike-in DNA) were added to each sample, and mixed with gentle vortexing. Samples were incubated for 10 min at 37°C, then centrifuged for 5 min at 4°C at 16,000 rpm. The tubes were placed on the magnetic stand and the supernatant was transferred to a clean 1.5 ml microcentrifuge tube. DNA extraction was performed utilizing standard phenol chloroform and ethanol precipitation methods as described (Skene et al. 2018). Samples were ethanol precipitated overnight at -20°C. DNA pellets were dissolved in 20 ul 0.1x TE Buffer (1 mM Tris-HCl pH 8, 0.1 mM EDTA). The Qubit High-Sensitivity Assay was used for DNA quantification.
Cut&Run libraries were prepared using the KAPA Hyper Prep Kit protocol, with some modifications. The total volume of Cut&Run DNA was used for library construction. For adapter ligation, 5 μl of 3 μM Adapter stock from the KAPA Dual-Indexed Adapter Kit was used. The ligation was incubated at 20°C for 15 min. The library was amplified using the following cycling conditions: 98°C - 45 sec, 98°C -15 sec, 60°C -10 sec, 14 cycles, 72°C for 1 min. After library amplification, the libraries were purified using 50 ul of KAPA Pure Beads and eluted in 20 μl of water.
mouse E13.5 ganglionic eminences were dissected, and single cell suspensions were processed for CUT&RUN, mouse E13.5 ganglionic eminences were dissected, and  processed for 4Cseq

value: mixed (C57/Bl6, 129Sv, FVB)


title: CTCF_0h_ChIPseq_Rep2


title: Puretype_3

type: RNA

city: Tokorozawa

first: Kosuke

last: Miyai

state: Saitama

address: Namiki 3-2

department: Basic Pathology

country: Japan

institute: National Defense Medical College

description: Gene expression of pure seminoma

hyb_protocol: 0.6 μμg of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 μμl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25 μμl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE 8x60K Microarray Ver3.0 (Agilent Technologies) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent Technologies) and 1 minute with 37°C GE Wash buffer 2 (Agilent Technologies).

scan_protocol: Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G2600D) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).

data_processing: The scanned images were analyzed with Feature Extraction Software (Agilent Technologies) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Processed signal intensities were normalized by the global scaling method. A trimmed mean probe intensity was determined by removing 2% of the lower and the higher end of the probe intensities in order to calculate the scaling factor. Normalized signal intensities were then calculated from the target intensity on each array using the scaling factor, so that the trimmed mean target intensity of each array was arbitrarily set to 2500.

source_name: Pure-type seminoma 3

label_protocol: Cyanine3 (Cy3) labeled cRNA was prepared from 0.1 μμg Total RNA using the Low Input Quick Amp Labeling Kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.

extract_protocol: Total RNA was isolated using the NucleoSpin totalRNA FFPE kit (Macherey-Nagel, Düren, Germany) in accordance with the manufacturer’s instructions. Samples with a minimal concentration of 50 ng/μl and DV200 50% or more, quantified using Agilent RNA 600 Nano kit (Agilent Technologies, Santa Clara, CA) and Agilent 2100 Bioanalyzer (Agilent Technologies), proceeded to further investigation.

treatment_protocol: For each case, we selected formalin-fixed, paraffin-embedded (FFPE) tissue blocks containing the areas in which histological diagnosis of seminoma was performed. These tissue blocks were then cut into 10-μm-thick sections and the tumor areas were manually microdissected into microcentrifuge tubes. The area of the cells microdissected from a single slide section typically ranged from 5 to 10 mm2.

value: Pure-type seminoma

tag: tissue preparation

value: FFPE block of resected tissue


title: PROseq_0h_R2

description:
No treatment; replicate #2
1698


title: 11 CUT&RUN IgG

source_name: Evf2TSTS E13.5GE


title: 10 CUT&RUN IgG


title: PIT_P1-PIT_P2-A9-AD008


title: PROseq_4h_R1

description:
4h auxin; replicate #1
1667


title: 200_ng_U005_SR


title: PIT_P3-PIT_P4-G10-AD006


title: PIT_P3-PIT_P4-G6-AD002


title: 200_ng_B3_IL

value: Illumina library prep


title: 8 CUT&RUN Dlx

value: anti-Dlx (Feng 2006)


title: 9 CUT&RUN Dlx


title: PROseq_0h_R1

description:
No treatment; replicate #1
1666


title: PIT_P3-PIT_P4-G8-AD010


title: 200_ng_B2_IL


title: 200_ng_B1_IL


title: PIT_P7-PIT_P8-F10-AD008


title: PIT_P7-PIT_P8-G9-AD007


title: 7 CUT&RUN Dlx


title: 6 CUT&RUN BRG1 (Smarca4)

value: anti-Brg1 (Wang 1996 )


title: 200_ng_U006_SR


title: 200_ng_A12_IL


title: PIT_P7-PIT_P8-D7-AD004


title: 5 CUT&RUN BRG1 (Smarca4)

value: E13.5 ganglionic eminence


title: 200_ng_A11_IL


title: PIT_P7-PIT_P8-G7-AD007


title: 4 CUT&RUN BRG1 (Smarca4)


title: 3 CUT&RUN Sox2

value: Bethyl Cat#A301-740A


title: 9v: WT

city: Orsay

first: Lionel

last: Larue

address: Centre Universitaire Bat 110

country: France

institute: Institut Curie

hyb_protocol: Complementary RNA (cRNA) was synthesized using the GeneChip 3′IVT Plus reagent Kit, according to the manufacturer’s protocol. Then 11μg cRNA were hybridized to mouse MOE430 2.0 gene expression Affymetrix microarrays.

scan_protocol: After washing and staining, using the Affymetrix fluidics station 450, the probe arrays were scanned using an Affymetrix GeneChip Scanner 3000.

data_processing: The dataset is normalized by the GCRMA procedure [1]  (quantile normalization) using custom cdf (chip  design le) based on Entrez from http://brainarray.mbni.med.umich.edu/Brainarray/Database/  CustomCDF/genomic_curated_CDF.asp (2). This cdf is build such that each gene is targeted by  a single probeset. (1) Diana. http://diana.cslab.ece.ntua.gr/. (2) Manhong Dai, Pinglang Wang, Andrew D Boyd, Georgi Kostov, Brian Athey, Edward G Jones,  William E Bunney, Richard M Myers, Terry P Speed, Huda Akil, Stanley J Watson, and Fan Meng.  Evolving gene/transcript de nitions signi cantly alter the interpretation of genechip data. Nucleic  Acids Res, 33(20):e175, 2005.

label: biotin

source_name: melanocyte cell line

label_protocol: Affymetrix protocol

growth_protocol: Melanocyte cell lines were grown up to 80% confluency in Ham’s F12 media supplemented with 10% FBS , antibiotics (100 U/mL penicillin and 100 ug/mL streptomycin), and 200nM TPA.

extract_protocol: Total RNA was extracted using the Allprep extraction kit (Qiagen) according to the manufacturer's instructions.

tag: genotype

value: WT

value: MALE

tag: rin

value: melanocyte cell line


title: 10_ng_U005_S

value: Adenocarcinoma (mammary gland), Melanoma, Hepatoblastoma (liver), Liposarcoma, Adenocarcinoma (cervix), Histiocytic lymphoma (macrophage; histocyte), Embryonal carcinoma (testis), Lymphoblastic leukemia(T lymphoblast), Glioblastoma (brain), Plasmacytoma/myeloma (B lymphocyte)

value: ThermoFisher (QS0639)

value: Swift library prep


title: Mouse Endometrium Individual 3

first: Katelyn

last: Mika

email: kmmika12@gmail.com, kmmika@uchicago.edu

state: IL

address: 1025 E. 57th Street

department: Organismal Biology; Genetic Medicine

institute: University of Chicago

data_processing:
Kallisto version 0.42.4 was used to pseudo-align the raw RNA-Seq reads to genomes
'kallisto index' with default parameters was used to build the genome index for each species
'kallisto quant' with the added parameters of 100 bootstraps, bias correction, and a flag for single end data with a !Sample_data_processing = fragment length of 50 and a standard deviation of 1 were used to quantify expression for each sample
Genome_build: Baboon (PapAnu2.0); Hamster (MesAur1.0); Bat (MyoLuc2.0); Squirrel (SpeTri2.0); Mouse (GRCm38)
Supplementary_files_format_and_content: Kallisto gene counts

source_name: pregnant mouse

extract_protocol:
Total RNA was extracted from the remaining tissue using the RNeasy Plus Mini Kit
Endometrial tissue samples from the pregnant uteri of baboon (x3), hamster (x3), bat (x2), squirrel (x2), and mouse (x3) were dissected and mailed to the University of Chicago in RNA-Later. These samples were further dissected to remove myometrium, luminal epithelium, and extra-embryonic tissues, and then washed 3x in ice cold PBS to remove unattached cell debris and red blood cells. Total RNA was extracted from the remaining tissue using the RNeasy Plus Mini Kit (74134, QIAGEN) per manufacturer’s instructions. RNA concentrations were determined by Nanodrop 2000 (Thermo Scientific). A total amount of 2.5μg of total RNA per sample was submitted to the University of Chicago Genomics Facility for Illumina Next Gen RNA sequencing. Quality was assessed with the Bioanalyzer 2100 (Agilent). A total RNA library was generated using the TruSEQ stranded mRNA with RiboZero depletion (Illumina) for each sample. The samples were fitted with one of six different adapters with a different 6-base barcode for multiplexing. Completed libraries were run on an Illumina HiSEQ2500 with v4 chemistry on 2 replicate lanes for hamster and 1 lane for other species, of an 8 lane flow cell, generating 30-50 million 50bp single-end reads per sample.

value: Pregnant endometrium

tag: molecule

value: rRNA depleted total RNA


title: PIT_P1-PIT_P2-C12-AD006

value: Pituitary

value: male


title: Mouse Endometrium Individual 2


title: PIT_P5-PIT_P6-A6-AD010


title: 13d: bcat*

value: bcat*

value: FEMALE


title: 14d: WT


title: PIT_P1-PIT_P2-D8-AD001


title: 10_ng_U003_S


title: Mouse Endometrium Individual 1


title: 2 CUT&RUN Sox2

value: mixed (C57/Bl6, 129Sv, FVB)


title: 10_ng_U004_S


title: 1 CUT&RUN Sox2


title: 10_ng_U002_S


title: C3H10T1-2_Setd2KO_H3K27ac

city: New York

first: Chao

last: Lu

state: New York

address: 1130 Saint Nicholas Ave

department: Genetics & Development

institute: Columbia University Medical Center

data_processing:
Basecalls performed using Illumina CASAVA version 1.7
For ATAC-seq, ChIP-seq and RNA-seq samples, Reads were aligned to the mm10 genome assembly using HISAT2 version 2.1.0 using default parameters
For ChIP-seq samples, ChIP-Rx normalization was performed according to Weinberg et al., Nature, 2019
For RNA-seq samples, Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
For WGBS samples, Reads were aligned to the mm10 genome assembly using BWA version 0.6.1 after converting the reference  genome to bisulfite mode
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files were generated using deepTools
Supplementary_files_format_and_content: tdf files were generated using IGVtools
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample ...

source_name: Cell line

extract_protocol:
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
ChIP-Rx libraries were prepared according to Weinberg et al., Nature, 2019.

value: embryo

value: fibroblast

tag: cell line

tag: genotype/variation


title: C3H10T1-2_Setd2KO_H3K4me1


title: parental_RNAseq_rep2

data_processing:
Basecalls performed using Illumina CASAVA version 1.7
For ATAC-seq, ChIP-seq and RNA-seq samples, Reads were aligned to the mm10 genome assembly using HISAT2 version 2.1.0 using default parameters
For ChIP-seq samples, ChIP-Rx normalization was performed according to Weinberg et al., Nature, 2019
For RNA-seq samples, Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using a protocol from Chepelev et al., Nucleic Acids Research, 2009. In short, exons from all isoforms of a gene were merged to create one meta-transcript. The number of reads falling in the exons of this meta-transcript were counted and normalized by the size of the meta-transcript and by the size of the library.
For WGBS samples, Reads were aligned to the mm10 genome assembly using BWA version 0.6.1 after converting the reference  genome to bisulfite mode
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files were generated using deepTools
Supplementary_files_format_and_content: tdf files were generated using IGVtools
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample.

extract_protocol:
RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: wildtype


title: parental_RNAseq_rep3


title: 10_ng_U001_S


title: C3H10T1-2_Parental_sgEZH2_K27ac


title: PIT_P7-PIT_P8-F11-AD007


title: parental_RNAseq_rep1


title: C3H10T1-2_H3K36M_RNAseq_rep3


title: PIT_P5-PIT_P6-F6-AD006


title: 100_ng_U020_SR

value: Swift Rapid library prep


title: PIT_P5-PIT_P6-F3-AD001


title: C3H10T1-2_Parental_sgCtrl_K27ac


title: PIT_P7-PIT_P8-C5-AD010


title: 100_ng_U021_SR


title: PIT_P5-PIT_P6-D2-AD001


title: C3H10T1-2_Setd2KO_RNAseq_rep3


title: C3H10T1-2_H3K36M_RNAseq_rep2


title: C3H10T1-2_Nsd1-2KO_EPZ6438_3uM_K27ac

value: Nsd1-2KO EPZ6438_3uM


title: 100_ng_U014_S


title: PIT_P3-PIT_P4-A12-AD002


title: PIT_P7-PIT_P8-B11-AD008


title: 10d: bcat*

value: MALE


title: PIT_P7-PIT_P8-E2-AD010


title: C3H10T1-2_Nsd1-2KO_RNAseq_rep2


title: 100_ng_U012_S


title: PIT_P7-PIT_P8-G1-AD012


title: C3H10T1-2_Setd2KO_RNAseq_rep2


title: C3H10T1-2_Nsd1-2KO_DMSO_K27ac

value: Nsd1-2KO DMSO


title: C3H10T1-2_H3K36M_RNAseq_rep1


title: C3H10T1-2_H3K36M_H3K4me1


title: PIT_P5-PIT_P6-H2-AD010


title: 100_ng_U013_S

value: Adenocarcinoma (mammary gland), Melanoma, Hepatoblastoma (liver), Liposarcoma, Adenocarcinoma (cervix), Histiocytic lymphoma (macrophage; histocyte), Embryonal carcinoma (testis), Lymphoblastic leukemia(T lymphoblast), Glioblastoma (brain), Plasmacytoma/myeloma (B lymphocyte)


title: 100_ng_U011_S

value: ThermoFisher (QS0639)


title: 100_ng_U019_SR


title: C3H10T1-2_H3K36M_H3K36me3


title: C3H10T1-2_Nsd1-2KO_H3K4me1


title: PIT_P1-PIT_P2-C10-AD001


title: C3H10T1-2_Nsd1-2KO_RNAseq_rep1


title: 100_ng_U004_SR


title: C3H10T1-2_H3K36M_H3K27ac


title: 100_ng_U015_S

value: Swift library prep


title: C3H10T1-2_Nsd1-2KO_RNAseq_rep3


title: PIT_P1-PIT_P2-B5-AD010


title: C3H10T1-2_Setd2KO_RNAseq_rep1


title: C3H10T1-2_Nsd1-2KO_H3K27ac


title: C3H10T1-2_H3K36M_sgEZH2_K27ac


title: PIT_P1-PIT_P2-C7-AD007


title: C3H10T1-2_H3K36M_sgCtrl_K27ac


title: PIT_P7-PIT_P8-C11-AD012


title: 100_ng_A8_IL

value: Illumina library prep


title: C3H10T1-2_Nsd1-2KO_ATAC

extract_protocol:
Cells were resuspended in 50uL of transposition reaction mix (2.5uL TDE1 Tn5 transposase and 1uL 0.5% Digitonin in 1x TD buffer) and incubated at 37°C for 30 min. The reaction was quenched using 1ul of 10% SDS and bead purification was performed using Agencourt AmpureXP beads (Beckman Coulter, cat # A63881) according to manufacturer instructions.
Transposition and library preparation were performed using the Nextera Kit (Illumina, cat # F-121-1030) and Index Kit (Illumina, cat # FC-121-1011). Tagmented DNA was amplified using i7 and i5 indexing adapters using a 5-cycle PCR program according to manufacturer instructions, size-selected using AmpureXP beads to remove species > 600 bp, and then normalized and pooled for sequencing.


title: PIT_P7-PIT_P8-C5-AD006


title: 100_ng_A7_IL


title: Tumor Treg pool 9

city: San Francisco

first: Stephen

last: Nishimura

address: 1001 Potrero Ave, Bldg 3, Rm211

department: Pathology

institute: UCSF

data_processing:
The computational pipeline for expression quantification is primarily based STAR aligner[1] and Cufflinks software tool [2].
Reads from RNA-seq were subjected to quality trimming using Trim_Galore (version 0.5.0) and aligned to mouse transcriptome (GRCm38.91) using STAR (version 2.6.1a) (Dobin et al., 2013). Duplicated reads were discovered using Picard tools and removed. Gene annotations (version GRCm38.91) were obtained from Ensembl.
TPM and FPKM values of genes were estimated using RSEM (version1.3.1) (Li and Dewey, 2011). “preprocessCore” (https://www.bioconductor.org/packages/release/bioc/html/preprocessCore.html).
FPKM values across the transcriptome and all samples were quantile normalized using the R package
Genome_build: mouse reference genome GRCm38.91
Supplementary_files_format_and_content: The quantile normalized FPKM values were used in downstream analyses.

source_name: Treg from b8 LLC + anti-b8, pool 3

extract_protocol:
RNA was isolated using the RNeasy Mini Kit (Qiagen) and processed as per manufacturer’s instructions.
cDNA synthesis and amplification was performed using the SMART-Seq Ultra® v4 Ultra® kit (TakaRa Bio USA, Inc.). The cDNA amplicons were quantified using Qubit (1X dsDNA High Sensitivity Assay) and analyzed with Agilent 2100 Bioanalyzer (High Sensitivity DNA Kit) for quality control. Sequencing libraries were constructed using the Nextera XT DNA Library Preparation Kit (Illumina, Inc.) from the cDNA.

treatment_protocol: Mice were treated 2 x/wk with anti-b8 (clone C6D4) (7 mg/kg I.P.) starting on day 7 after tumor innoculation. Tumors were harvested on day 14.

tag: background strain

value: wild-type

value: Treg

value: anti-b8 (C6D4)


title: PIT_P3-PIT_P4-H10-AD001

value: Pituitary

value: male


title: Tumor Treg pool 8

source_name: Treg from b8 LLC + anti-b8, pool 2


title: Tumor Treg pool 7

source_name: Treg from b8 LLC + anti-b8, pool 1


title: C3H10T1-2_Parental_ATAC

value: parental


title: 100_ng_A6_IL


title: PIT_P7-PIT_P8-C4-AD010


title: PIT_P7-PIT_P8-G4-AD006


title: PIT_P5-PIT_P6-B5-AD001


title: 100_ng_A10_IL


title: Tumor Treg pool 6

source_name: Treg from b8 LLC + isotype, pool 3

value: isotype


title: 100_ng_U003_SR


title: GABAergic interneuron -AAV-shbrpf1 1

city: Shanghai

first: Jingli

last: Cao

state: Shanghai

address: 131 Dongan Road, Xuhui District, Shanghai

country: China

institute: Fudan university

data_processing:
Illumina Casava1.7 software used for basecalling.
The Skewer software was used to dynamically remove the adapter sequence fragments and low-quality fragments from the 3'end of the sequencing data, and the FastQC software was used to perform quality control analysis on the preprocessed data and count the base ratios of Q20 and Q30.
For all samples, StringTie software was used to count the original sequence counts of known genes, and the expression of known genes was calculated using FPKM-Fragments Per Kilobase of transcript per Million fragments mapped.The DESeq2 software was used to screen the differentially expressed genes between different sample groups, and the differential expression range of |log2FC| >= 1 and Pvalue <= 0.05 was used to screen the differentially expressed genes between the two groups.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample ...

source_name: Neural progenitor cells

growth_protocol: Primary GABAergic interneurons were prepared from E14.5 wild-type embryonic brain，plated at a density of 2-5 × 104 cells / well on coverslips coated with poly-L-lysine (Sigma, P9155) in advance in 24-well plate, and cultured on a neurobasal medium supplemented with 2% B27 supplement, 1% GlutaMAX-1.

extract_protocol:
RNA was harvested using Trizol reagen,dissolved it in a suitable volume of RNase-free water, and determined the RNA concentration with a spectrophotometer.
RNA libraries were prepared for the paired-end sequencing mode of the Illumina Hiseq sequencing platform to perform high-throughput sequencing on multiple samples

treatment_protocol: Adeno-associated virus infection for 12 hours

tag: development stage

value: embryonic day 14.5

value: Adeno-associated virus infection

value: Nerve tissue


title: Tumor Treg pool 5

source_name: Treg from b8 LLC + isotype, pool 2

tag: cell type


title: GABAergic interneuron -AAV-shbrpf1 3

tag: strain


title: Tumor Treg pool 4

source_name: Treg from b8 LLC + isotype, pool 1


title: GABAergic interneuron -AAV-shbrpf1 2


title: C3H10T1-2_H3K36M_ATAC

value: embryo

value: fibroblast


title: PIT_P3-PIT_P4-A5-AD002


title: 100_ng_A9_IL

tag: commercial source


title: HT144_Combo_Rep1

city: Camperdown

first: Stuart

middle: J

last: Gallagher

state: NSW

address: Building 93, RPA Hospital

department: Melanoma Oncology

country: Australia

institute: Centenary Institute

data_processing:
Fastq files were checked for quality using Fastqc
Reads were aligned to Human-GRCh38-93 using STAR version 020201, from the Subread package. The genomeGenerate command in STAR was used to build the genome prior to alignment. STAR aligment produced unsorted bam files
BAM files were sorted using the "sort" command in samtools version 1.3.1
Aligned and sorted BAM files were then counted into features at the gene level using featureCounts version 1.5.0-p3 with options "-t exon -g gene_id -T 12 -a" to generate the featureCounts_BETCDK9i.txt file
Library-size normalised data was calculated using the counts(df, normalized = TRUE) command in the DeSEQ2 package version 1.26.0 in R version 3.5.3
Genome_build: Human-GRCh38-93
Supplementary_files_format_and_content: featureCounts_BETCDK9i.txt is counts of the number of fragments  per gene, generated from using the featureCounts tool as above.This data is un-normalised.
Supplementary_files_format_and_content: rlog_values_CDK9_RNAseq.csv is Regularized log transformed gene expression values using the DESEQ2 "rlog" function.

source_name: cultured melanoma cells

growth_protocol: Melanoma cells (10^6) were seeded in a T75 in RPMI. The next day cells were treated with the appropriate drug and harvested 24 h later.

extract_protocol:
Total RNA was extracted using RNeasy Mini Plus Kits (Qiagen, Hilden, Germany) with cells being lysed directly in culture flasks
A sequencing library was prepared with NEBNext Ultra RNA Library Prep Kit for Illumina, and sequenced on an Illumina NovaSeq6000, 100bp single end reads.

treatment_protocol: Cells were treated for 24 h with either DMSO (carrier control), 2μM IBET151, 200nM CDKI73 or the combination of both drugs.

value: melanoma

value: Combo

tag: biological replicate


title: HT144_I73_Rep2


title: Tumor Treg pool 3

source_name: Treg from Mock LLC, pool 3

value: untreated


title: C3H10T1-2_Setd2KO_ATAC


title: PIT_P3-PIT_P4-C5-AD002


title: HT144_I73_Rep1


title: Tumor Treg pool 2

source_name: Treg from Mock LLC, pool 2


title: HT144_IBET151_Rep2


title: GABAergic interneuron -AAV-scramble 1


title: Tumor Treg pool 1

source_name: Treg from Mock LLC, pool 1


title: PIT_P5-PIT_P6-D11-AD004

source_name: C57BL/6J Male P56 Mouse Brain


title: HT144_Combo_Rep2


title: R586W clone 2

city: Chapel Hill

first: Zachary

last: Carico

state: NC

address: 250 Bell Tower Dr, CB7100

department: Integrative Biological and Genome Sciences program

institute: University of North Carolina

data_processing:
Initial data processing, including merging of replicates, alignment, filtering, and generation of Hi-C contact maps, was perfomed using the Juicer wrapper from the Lieberman-Aiden lab.
Contact domains were called from merged, Knight-Ruiz balanced contact maps at 10kb resolution using the Arrowhead algorithm with default parameters.
Loops were called from merged, Knight-Ruiz balanced contact maps at 5kb, 10kb, and 25kb resolution using the HiCCUPs algorithm with default parameters except: -p 8,4,2 -i 14,10,6
Genome_build: mm10
Supplementary_files_format_and_content: Hi-C matrices are provided in .hic format. Filtered Hi-C contacts are provided in .txt format. Loop and domain calls are provided in .bedpe format.

source_name: Mouse embryonic stem cells v6.5

growth_protocol: V6.5 murine embryonic stem cells (mESCs) (male) were grown under standard conditions, as previously described (Dowen et al, 2014)

extract_protocol:
Hi-C libraries were prepared using the Arima Hi-C kit (Arima Genomics) according to manufacturer's specifications, with one modification. Treatment with DNA ligase was performed for one hour instead of the recommended 15 minutes.
Dual-indexed Illumina sequencing libraries were prepared using Kapa Hyper Prep (Kapa Biosciences, cat.#KK8502) according to specifications provided by Arima Genomics, with one modification. Ligation of sequencing adapters was allowed to proceed for 1 hour instead of the recommended 15 minutes.

treatment_protocol: ESCs growing in exponential phase were placed in single cell suspension in 1x PBS with 1% formaldehyde. After 2 minutes, crosslinking was quenched with addition of glycine to 150mM

value: mESC v6.5


title: PIT_P5-PIT_P6-A1-AD002


title: WT rep2

value: Wild Type


title: BT added high-fat diet_mRNA [L_BT_2]

city: Gwangju

first: Ji-Young

last: Choi

address: 30, Chosundae 3-gil, Dong-gu

department: Food and Nutrition

country: South Korea

institute: Chosun University

data_processing:
Illumina CASAVA 1.8.2 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence and low-quality sequence
Mapped to M. musculus whole genome using STAR v2.4.0
Transcripts were assembled and quantitated using Cufflinks v2.1.1
Genome_build: M. musculus Ensembl 72
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for "HFD" and "BT" samples.

source_name: BT added high-fat diet_liver

growth_protocol: Twenty male C57BL/6J mice (four-week-old, male) were obtained from the Jackson Laboratories (Bar Harbor, ME, USA). All mice were individually housed under a constant temperature (24°C) and 12-h light/dark cycle and, fed a normal chow diet for acclimation for 1 week after arrival.

extract_protocol:
Total RNA was extracted from the liver using TRIZOL reagent (Invitrogen Life Technologies, NY) according to the manufacturer’s instructions. Quality control was performed with Agilent Bioanalyser.
Libraries were constructed from mRNA using the Illumina TruSeq RNA Sample Prep Kit following  manufacturer's protocols.

treatment_protocol: At 5 weeks of age, they were randomly divided into 2 groups of 10 mice per each group and fed a high-fat diet (HFD), HFD + 1.5% (w/w) Bangpungtongseong-san extract BT) for 12 weeks

value: 17 weeks

value: high-fat diet (HFD) + 1.5% (w/w) Bangpungtongseong-san extract BT) for 12 weeks

value: liver


title: HT144_IBET151_Rep1


title: BT added high-fat diet_mRNA [L_BT_1]

value: high-fat diet (HFD) + 1.5% (w/w) Bangpungtongseong-san extract for 12 weeks


title: BT added high-fat diet_mRNA [L_BT_3]

value: male


title: HT144_DMSO_Rep2

value: DMSO


title: H3K27me3 WT rep 2

data_processing:
Alignment:  reads were aligned to a hybrid mm10+hg38 genome with  bowtie v 1.2.2 with parameters -v 2 -p 24 -S -m 1 --best --strata
PCR Duplicate Removal: duplicates removed with samtools v 1.9 fixmate -m followed by markdup -r -s
Peak Calling: peaks were called using MACS (v 2016-02-15) with parameters -f BED -g mm -q 0.01
Coverage File Generation: bigwig files were generated using bedtools (v 2.26) genomecov with parameters -bga -g mm10.chrom.sizes followed by ucsctools (v 320) bedGraphToBigWig
Genome_build: mm10
Supplementary_files_format_and_content: For each ChIPped factor, a Z-score-normalized BigWig coverage track of merged replicates is provided for viewing in UCSC genome browser.

growth_protocol: V6.5 murine embryonic stem cells (mESCs) (male) were grown in media supplemented with FBS and LIF under standard conditions, as previously described (Dowen et al, 2014)

extract_protocol:
Cells were crosslinked in 1% formaldehyde for 5 minutes, and lysed using sequential treatment with hypotonic and detergent-containing buffers. Chromatin was sheared to 200-1000bp using a Covaris sonicator.
Kapa Hyper Prep library kit was used according to manufacturer's instructions.

treatment_protocol: mESCs were crosslinked in 1% formaldehyde (ThermoFisher, 28906) for 5 minutes in PBS and quenched by adding glycine. Cells were lysed using Lysis Buffers 1 and 2, and recovered nuclei resuspended in 1ml Covaris Sonication Buffer (50mM Tris pH 7.5, 10mM EDTA, 0.1% SDS). Nuclei were sheared for 12 minutes using a Covaris E220 water bath sonicator with device settings of duty factor 5, PIP/W of 140, and 200 cycles per burst. Insoluble material was cleared by spinning sonicated lysates for 10 minutes at 21,000rcf. 10µg  antibody  was incubated for 8 hours with 30µl of Dynabeads in PBS, after which excess antibodies were washed from the beads. 10^7 cell equivalents of sheared chromatin were incubated overnight with the antibody-bound beads in 1 ml Sonication Buffer 1, washed with Wash Buffers 1, 2, and 3, and crosslinks were reversed overnight. DNA was purified using a ChIP DNA Clean and Concentrator Kit (Zymo, D5201) according to manufacturer’s instructions. Sequencing libraries were prepared using a Hyper Prep kit (Kapa Biosciences, KK8502) according to manufacturer’s instructions, and 50bp single-end sequencing reads were collected using the Illumina NovaSeq sequencing platform and reagents.

tag: antibody

value: abcam ab6002


title: PIT_P1-PIT_P2-C7-AD004

value: Pituitary


title: H3K27ac WT rep 3

source_name: Mouse embryonic stem cells v6.6

value: mESC v6.6

value: Active Motif 39136


title: PIT_P3-PIT_P4-H11-AD010


title: High-fat diet_mRNA [L_HFD_3]

source_name: High-fat diet_liver

tag: treatment

value: high-fat diet (HFD) for 12 weeks


title: GABAergic interneuron -AAV-scramble 3

value: embryonic day 14.5

value: Adeno-associated virus infection

tag: tissue

value: Nerve tissue


title: GABAergic interneuron -AAV-scramble 2


title: HT144_DMSO_Rep1

value: melanoma


title: C057_Combo_Rep2

value: Combo


title: High-fat diet_mRNA [L_HFD_2]

tag: age


title: C057_Combo_Rep1


title: PIT_P7-PIT_P8-C8-AD004

tag: Sex


title: H3K4me3 R586W clone 2 rep 2

value: SMC1A-R586W clone 2

value: mESC v6.5

value: abcam ab8580


title: PIT_P7-PIT_P8-A9-AD002


title: PIT_P3-PIT_P4-F3-AD001


title: High-fat diet_mRNA [L_HFD_1]

value: 17 weeks

value: liver


title: C057_I73_Rep2


title: PIT_P3-PIT_P4-G11-AD007

value: male


title: H3K4me3 R586W clone 1 rep 2

value: SMC1A-R586W clone 1


title: PIT_P7-PIT_P8-B7-AD008


title: H3K4me3 R586W clone 1 rep 1


title: PIT_P1-PIT_P2-E10-AD012


title: H3K4me3 WT rep 1

value: Wild Type


title: PIT_P1-PIT_P2-A5-AD006

value: Pituitary


title: C057_DMSO_Rep2

value: DMSO


title: C057_IBET151_Rep2


title: C057_IBET151_Rep1


title: PIT_P1-PIT_P2-B3-AD008


title: C057_I73_Rep1


title: H3K4me3 WT rep 2


city: Rochester

first: Wenqian

last: Hu

state: Minnesota

address: 200 First St SW, GU16-01A

department: Biochemistry and Molecular Biology

institute: Mayo Clinic

description:
small RNA
small RNA-seq in the CLIP_deletion mESC, biological replicate 4, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv

data_processing:
Basecalls performed using CASAVA version 1.4
For RNAseq and CLIPseq, sequenced reads were trimmed for adaptor sequence, then mapped to mm10 whole genome using hisat2 with parameters--mp 6,2. The resulting sam file is converted into bam file using samtools. The uniquely mapped reads were isolated using samtools.
For small RNAseq, sequenced reads were trimmed for adapter sequence using cutadapt. The resulting reads with a length between 17 and 30nt were retained for mapping by HISAT2 (v2.1.0).
The differentially expressed and non-differentially expressed miRNAs were identified by EdgeR using the Negative Binomial Generalized Linear Models with Quasi-Likelihood Tests.
The CLIPseq peak calling were performed by the Piranha, iCount, and CLIPper. The common peaks from these 3 programs were retained as Trim71-binding regions
Genome_build: mm10
Supplementary_files_format_and_content: csv files containing miRNA expression levels (in TPM) from the smallRNA-seqs, gene expression levels (in TPM) from the RNAseqs , and Trim71-binding regions from the CLIPseq

source_name: mouse embryonic stem cells

growth_protocol: All the mESCs used in this study were cultured on 0.5% gelatin-coated tissue culture plates in either the 15% + Lif medium (DMEM/F-12 supplemented with 15% FBS, 2mM L-Glutamine, 0.1mM MEM NEAA, 1% Penicillin-Streptomycin, 0.1 mM β-mercaptoethanol and 1000 U/mL mLIF) or the 2i + Lif medium (DMEM/F-12, 2% FBS, 2mM L-Glutamine, 0.1mM MEM NEAA, 1% Penicillin-Streptomycin, 0.1 mM β-mercaptoethanol and 1000 U/mL mLIF, 1 x N2N27, 3 uM CHIR99021 and 1 uM PD0325901).

extract_protocol:
Total RNA were isolated from mESCs via Trizol reagent
RNA-seq library was constructed via the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (E7420S). The small-RNA-seq libraries were prepared using the NEBNext Multiplex Small RNA Library Prep Set for Illuminia (Set 1) (E7300S). The Trim71 CLIP-seq was performed using the previously established HITS-CLIP protocol (Moore et al., 2014).
CLIP-seq, RNA-seq, small RNA-seq

value: the CLIP deletion mESCs

tag: genetic background


title: PIT_P1-PIT_P2-A6-AD001


title: PIT_P1-PIT_P2-B4-AD001


description:
small RNA
small RNA-seq in the CLIP_deletion mESC, biological replicate 3, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: C002_Combo_Rep1


title: H3K4me3 R586W clone 2 rep 1

tag: genotype


title: C057_DMSO_Rep1


title: C002_Combo_Rep2


description:
small RNA
small RNA-seq in the CLIP_deletion mESC, biological replicate 2, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: PIT_P7-PIT_P8-E1-AD007


title: C002_I73_Rep2


description:
small RNA
small RNA-seq in the CLIP_deletion mESC, biological replicate 1, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: PIT_P3-PIT_P4-E11-AD006


title: C002_I73_Rep1


title: C002_IBET151_Rep2


description:
small RNA
small RNA-seq in the wild-type mESC, biological replicate 4, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv

value: the wild-type mESCs


description:
small RNA
small RNA-seq in the wild-type mESC, biological replicate 3, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: C002_DMSO_Rep2


title: MM_Pit_4-2 [snRNA-seq]

city: Los Angeles

first: Michel

middle: A

last: Zamojski

state: California

address: 8700 Beverly Boulevard

department: Regenerative Medicine Institute

institute: Cedars-Sinai Medical Center

description: nuclear mRNA

data_processing:
sn-RNAseq data were processed using the Cell Ranger pipeline v3.0.2.
Snap-frozen samples were aligned to a whole transcripts pre-mRNA reference transcriptome.
The pre-mRNA reference package was generated from the mm10 reference transcriptome using Cell Ranger mkref such as to include alignments to whole, unspliced, transcripts.
Dissociated samples were aligned to the standard mm10 reference transcriptome
Genome_build: mm10
Supplementary_files_format_and_content: UMI count matrices in Hierarchical Data Format HDF5 for cell barcodes

source_name: Pituitary gland

growth_protocol: Animals were on a 12-hour on, 12-hour off light cycle, with lights on at 7 AM and off at 7 PM. Pituitaries were collected from male and randomly-cycling C57BL/6 female mice aged 10-12 weeks.

extract_protocol:
ii) Snap-frozen pituitaries were thawed on ice. For nuclei isolation, a modified protocol from 52 was followed, all on ice. RNAse inhibitor (NEB MO314L) was added to the homogenization buffer (0.32 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 5mM CaCL2, 3mM Mg(Ac)2, 0.1% IGEPAL CA-630), 50% OptiPrep (Stock is 60% Media from Sigma; cat# D1556), 35% OptiPrep and 30% OptiPrep right before isolation. Each pituitary was homogenized with 10 strokes (loose pestle) in an individual autoclaved dounce glass homogenizer (1ml, VWR cat# 71000-514). The homogenate was filtered through a 40-micrometer cell strainer. The volume was measured and an equal volume of 50% OptiPrep was added. The gradient was centrifuged using an SW41 rotor at 9200 rpm and at 4C for 25 min. Nuclei were collected from the interphase between 30% and 35% OptiPrep. The nuclei were washed, resuspended either in 1x nuclei dilution buffer for snATACseq (10X Genomics) or in 1X PBS/0.04% BSA for snRNAseq, and counted on an Invitrogen Countess II cell counter. ii) Dissociated pituitary cells were cryopreserved in 90% FBS/10% DMSO and shipped to New York on dry-ice. Upon arrival, cells were stored in liquid nitrogen. On the day of an experiment, the vial was removed from liquid nitrogen on dry-ice, thawed quickly in a 37C waterbath, and cells were serially diluted with 10% FBS /DMEM until a minimum volume of 8ml was reached. Cells were centrifuged at 300xg for 5min, resuspended in 1ml of medium and counted. Nuclei were isolated from all cells remaining after the scGEM Drop-seq RNA-seq assay was done. Cells were centrifuged at 500g for 5min at 4C. The supernatant was carefully removed and 0.1X lysis buffer (1x: 10mM Tris-HCL pH 7.5, 10mM NaCl, 3mM MgCl2, nuclease-free H2O, 0.1% v/v NP-40, 0.1% v/v Tween-20, 0.01% v/v digitonin) was added. After 5min incubation on ice, 1ml of wash buffer was added. The nuclei were pelleted at 500xg for 5min at RT, resuspended either in 1X PBS/0.04% BSA for scGEM Drop-seq RNA-seq or in diluted nuclei buffer for snATAC-seq. Nuclei were counted and the concentration adjusted to run the given assay.
snRNAseq were performed as described (10x Genomics, Pleasanton, CA; (24)), following the Single Cell 3’ Reagents Kits V3 User Guidelines. Nuclei were filtered and counted on a Countess instrument. The amount of nuclei loaded on the chip was determined based on the 10X Genomics protocol. The 10X chip (Chromium Single Cell 3’ Chip kit A v2 PN-12036 or v3 chip kit B PN-2000060) was loaded to target a minimum of 1,000 nuclei final. Reverse-transcription was performed in the emulsion and cDNA was amplified before library construction following the Chromium protocols v2 or v3. Each library was tagged with a different index for multiplexing (Chromium i7 Multiplex kit PN-12062). Quality control and quantification of the amplified cDNA were assessed on a Bioanalyzer (High-Sensitivity DNA Bioanalyzer kit). Sequencing was carried out at the NYGC on Illumina Novaseq using 98+26 paired-end reads.

treatment_protocol: i) Snap-frozen pituitaries: pituitaries were immediately snap-frozen following dissection, and stored at -80C. ii) Dissociated pituitaries: upon collection, pituitaries were digested with collagenase and dispersed with pancreatin and the dissociated cells collected acutely. About 0.7-2.5x10^6 cells were collected from single pituitaries and placed in individual tubes for cryopreservation.

value: 10-12 weeks

tag: source type

value: dissociated, cryopreserved pituitaries

value: Male


title: 2_cell transfected siFra1+siFra2-rep3

city: Montpellier

first: Isabelle

last: Jariel-Encontre

state: Montpellier Cedex5

address: 208 Avenue des Apothicaires

institute: IRCM-U1194- INSERM

hyb_protocol: cDNA targets were used to probe Affymetrix GeneChip Human Gene 2.0 ST arrays, which were then washed, stained and scanned according to Affymetrix instructions (user manual P/N 702731 Rev. 3)

scan_protocol: GeneChips were scanned using GeneChip Scanner 3000

data_processing: 21 CEL (7 conditions, n=3 each) files generated after array scanning were imported into the Partek Genomics Suite 6.6 (Partek) for preprocessing (default Partek options)

label: Biotin

label_protocol: biotinylated sense-strand cDNA were produced using the GeneChip WT PLUS Reagent kit from Affymetrix according to the manufacturer’s specifications

growth_protocol: MDA-MB-231 cells were cultured in in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and penicillin/streptomycin (100µg/ml each)

extract_protocol: RNAs were extracted using the Qiagen miRNeasy kit according to the supplier's specifications and controlled for purity and integrity using the Agilent Bioanalyser with RNA600 nano LabChipkits (Agilent technologies). On-column DNAse I digestions were performed as described in the user’s guide. RNA with neither signs of contamination nor of degradation (RIN>9) were further processed to generate aplified and biotinylated sense-strand cDNA

tag: batch

value: T2- transcriptome 2

tag: replicate

value: C


title: FM_Pit_2-2 [snRNA-seq]

description: nuclear mRNA

value: Female


description:
small RNA
small RNA-seq in the wild-type mESC, biological replicate 2, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: FrozPit-FM3 [snRNA-seq]

description: nuclear pre-mRNA

value: snap-frozen pituitaries


title: C002_IBET151_Rep1

tag: cell line

value: melanoma


title: FM_Pit_1-2 [snRNA-seq]

description: nuclear mRNA


title: 2_cell transfected siCTL-rep1

value: siCTL

value: A


title: small RNA-seq in the Ago2 deletion and CLIP deletion mESC, biological replicate 4

description:
small RNA
small RNA-seq in the Ago2_deletion_and_CLIP_deletion mESC, biological replicate 4, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv

value: Ago2 deletion and CLIP deletion mESCs


description:
small RNA
small RNA-seq in the wild-type mESC, biological replicate 1, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: C002_DMSO_Rep1


title: 2_cell transfected siCTL-rep3


title: FrozPit-FM2 [snRNA-seq]

description: nuclear pre-mRNA


title: 2_cell transfected siCTL-rep2

value: B


title: MDA-MB-231: p300/CBP in cells transfected with siRNA directed against Fra-1

data_processing:
Reads were aligned to hg19 genome using Bowtie2 allowing no more than 2 mismatches and no multiple alignment sites.
Aligned reads were processed with R package PASHA (Fenouil et al., 2013; doi: 10.1093/bioinformatics/btw206) to eliminate PCR artefacts, extend reads to an estimated fragment length and generate WIG files.
R Package PASHA was used to normalize the WIG files to calculate the scores per million reads sequenced and Input DNA signal was subtracted from normalized WIG files.
Peaks were called using Thresholding function of Integrated Genome Browser (https://bioviz.org/) with the following settings : threshold=25, Max gap=50, Min run=50
Genome_build: Hg19
Supplementary_files_format_and_content: bigwig files

source_name: MDA-MB-231 cell line

growth_protocol: MDA-MB-231 cells (ATCC, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and penicillin/streptomycin (100µg/ml each) in a humidified 5% CO2 atmosphere at 37°C. Cells freshly amplified and frozen after obtention from the ATCC were used every 3months and were routinely tested for the absence of mycoplasma contamination

extract_protocol:
Lysates were clarified from sonicated nuclei and ChIP were carried out with the anti-p300/CBP antibody from Santa Cruz Biotechnology, sc-32244X.
ChIP-seq libraries were prepared using the TruSeq ChIP sample preparation kit from Illumina by the MGX sequencing Platform (Montpellier, France)

treatment_protocol: MDA-MB-231 cells were transfected with 4.5 nM of a control siRNA (Silencer® Negative Control N°1 siRNA, Ambion # AM4611) or 4.5 nM of a mix of 3 siRNA directed against Fra-1 (Tolza et al. 2019, Mol Cancer Research, DOI:10.1158/1541-7786.MCR-19-0036) for 72h before fixation in 1% paraformaldehyde for 7 min at 25°C.

value: breast cancer cell line

value: triple negative - basal b


title: small RNA-seq in the Ago2 deletion and CLIP deletion mESC, biological replicate 3

description:
small RNA
small RNA-seq in the Ago2_deletion_and_CLIP_deletion mESC, biological replicate3, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: 2_cell transfected siFra1+siFra2-rep1


title: FrozPit-FM1 [snRNA-seq]

description: nuclear pre-mRNA


title: MDA-MB-231: p300/CBP in cells transfected with control siRNA


title: MDA-MB-231: Input


title: 2_non transfected cells-rep3

value: NT


title: FrozPit-MM3 [snRNA-seq]

description: nuclear pre-mRNA


title: small RNA-seq in the Ago2 deletion and CLIP deletion mESC, biological replicate 1

description:
small RNA
small RNA-seq in the Ago2_deletion_and_CLIP_deletion mESC, biological replicate 1, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: small RNA-seq in the Ago2 deletion and CLIP deletion mESC, biological replicate 2

description:
small RNA
small RNA-seq in the Ago2_deletion_and_CLIP_deletion mESC, biological replicate 2, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: small RNA-seq in the Ago2 deletion mESC, biological replicate 4

description:
small RNA
small RNA-seq in the Ago2_deletion mESC, biological replicate 4, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv

value: Ago2 deletion mESCs


title: 2_cell transfected siFra1+siFra2-rep2


title: 2_non transfected cells-rep1


title: 2_non transfected cells-rep2


title: V4_post

city: Berlin

first: Helene

last: Kretzmer

address: Ihnestraße 63

department: Genome Regulation

country: Germany

institute: Max Planck Institute for Molecular Genetics

description: RRBS

data_processing:
Bulk RRBS processing: Bisulfite-converted reads were aligned to the hg19 reference genome with bsmap using the following parameter: -v 0.05 -s 16 -w 100 -S 1 -R -u -m 0. Methylation rates were called using custom scripts and subsequently filtered for coverage (min 10).
Single cell processing: Adapter clipping and quality trimming using cutadapt. Bisulfite-converted reads were aligned to the hg19 reference genome with bsmap using the following parameter: -v 0.1 -s 12 -q 20 -w 100 -S 1 -R -u -m 0. Methylation rates were called using mcall.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig file from coverage filtered Bed file (<chr> <start> <end> <methylation rate>)

source_name: blood

extract_protocol:
Genomic DNA for RRBS library generation was isolated and quantified using previously described methods (Boyle et al., 2012)
RRBS library generation was performed as previously described (Boyle et al., 2012). Genomic DNA was enzymatically sheered using MspI endonuclease (New England Biolabs, catalogue number R0106L), which exposes a CpG site at the 3’-end. Barcoded Illumina sequencing adapters were added to each end of the DNA strand. The DNA was then subjected to two rounds of sodium bisulfite conversions using the Epitect Bisulfite Conversion Kit (Qiagen, catalogue number 59104). A final, large-scale PCR was conducted on the converted DNA (which uracil is replaced by thymine) and the product was submitted for sequencing.

tag: patient id

tag: diagnosis

value: CLL

tag: ighv

value: Unmutated

tag: treatmentstatus

value: post-treatment


title: shWDR5_H3K4ac_CUT&Tag

city: Jinan

first: Tianli

last: Chen

state: Shandong

address: 107 West Wenhua Road

department: Department of General Surgery

institute: Qilu Hospital of Shandong University

description: Knockdown of WDR5 using shRNA

data_processing:
Library strategy: CUT&Tag
Libraries were sequenced on Novaseq(Illumina, USA) platform and 150-bp paired-end reads were generated.
Fastp software was used to remove adapter and low-quality reads.
All reads produced by CUT&Tag of H3K4ac were aligned to the hg19 human genome using BWA(Burrows Wheeler Aligner).
MACS2 was used for peak calling. Genes with a peak within 3 kb of the TSS were considered target genes.
Annotation of peaks was performed using ChIPseeker.
Genome_build: hg19
Supplementary_files_format_and_content: txt files include peaks of each sample

source_name: human cholangiocarcinoma cell

growth_protocol: QBC939 was cultured in DMEM (Thermo Fisher Scientific). The media was supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C under 95% air and 5% CO2.

extract_protocol:
CUT&Tag(Cleavage Under Targets and Tagmentation) Kit for Illumina was purchased from Vazyme(TD901). The assay was performed according to the protocol. Briefly, 1 × 105 QBC-939 cells were washed twice with wash-buffer and incubated with Concanavalin A-coated magnetic beads (ConA beads) at room temperature for 15 min. Bead-bound cells were collected by magnet stand and resuspended in 50 μl of antibody-buffer. 1 μg of anti-H3K4ac antibody (39381, Active Motif) was added and incubated at room temperature for 2 hours with slow rotation. 1 μg secondary antibody(goat anti-rabbit IgG H&L, Ab206, Vazyme) was diluted in 50 μl of Dig-wash buffer and cells were incubated at room temperature for 50 min. The Hyperactive pG-Tn5 Transposon was diluted 1:100 in Dig-300 buffer and incubated with cells at room temperature for 1 h. Cells were then resuspended in Tagmentation buffer for DNA fragmentation. After 1 hour,  EDTA, SDS and Proteinase K were added to terminate tagmentation. DNA was then purified using phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation.
Library amplification was also performed according to the protocol. 24 μl DNA was mixed with 1 μl TAE, 10 μl 5×TAB, 5 μl ddH2O as well as 5 μl i5 and i7 primers from TruePrep Index Kit V2 for Illumina (Vazyme, TD202). PCR program was set according to the protocol. VAHTS DNA Clean Beads (Vazyme) was used for DNA purification. Libraries were sequenced on Novaseq(Illumina, USA) at Novogene Science and Technology CO.,Ltd(Beijing,China) and 150-bp paired-end reads were generated. Fastp software was used to remove adapter and low-quality reads. All reads produced by CUT&Tag of H3K4ac were aligned to the hg19 human genome using BWA(Burrows Wheeler Aligner). MACS2 was used for peak calling. Genes with a peak within 3 kb of the TSS were considered target genes. An area of 6 kb surrounding each TSS was selected to get CUT&Tag profiles of H3K4ac using deepTools software. Annotation of peaks was performed using ChIPseeker.

treatment_protocol: WDR5 was knocked down in QBC939 cells with shRNA.

tag: cell lines

value: QBC-939 cells

tag: first antibody

value: H3K4ac (Active Motif，39381)

tag: shRNA


title: FrozPit-MM2 [snRNA-seq]

description: nuclear pre-mRNA

tag: strain

value: 10-12 weeks

value: Male


title: cell transfected siFra-2-rep3

value: T1- transcriptome 1

tag: treatment

value: C


title: cell transfected siFra-1-rep3


title: small RNA-seq in the Ago2 deletion mESC, biological replicate 3

description:
small RNA
small RNA-seq in the Ago2_deletion mESC, biological replicate 3, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv

tag: cell type


title: small RNA-seq in the Ago2 deletion mESC, biological replicate 2

description:
small RNA
small RNA-seq in the Ago2_deletion mESC, biological replicate 2, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv


title: V4_pre

description: RRBS

value: pre-treatment


title: cell transfected siFra-2-rep2


title: cell transfected siFra-2-rep1

value: A


title: cell transfected siCTL-rep3

value: siCTL


title: scramble_H3K4ac_CUT&Tag

description: scramble

value: scramble


title: FrozPit-MM1 [snRNA-seq]

description: nuclear pre-mRNA

tag: age

value: snap-frozen pituitaries

tag: Sex


title: Male_Pit_4

data_processing:
Single Nuclei ATAC-seq data of dissociated pituitary samples were processed using Cell Ranger-ATAC pipeline version 1.1.0 with mm10 as the reference genome.
The data was then cleaned of barcode multiplets using a privately distributed python script from 10X Genomics. (clean_barcode_multiplets_1.1.py)
Single Nuclei ATAC-seq data of snap-frozen pituitary samples were processed using Cell Ranger-ATAC pipeline version 1.2.0 with mm10 as the reference genome.  Version 1.2.0 properly removes barcode multiplets.  Thus, no additional processing was needed.
Genome_build: mm10
Supplementary_files_format_and_content: Peak accessibility count matrices in Hierarchical Data Format HDF5 for cell barcodes.
Supplementary_files_format_and_content: Metadata files that include per barcode QC, ATAC signal and cell calling metrics in comma-separated values (CSV) format.
Supplementary_files_format_and_content: BED-like tabular files listing all unique fragments captured by the assay in tab-separated values (TSV) format.

extract_protocol:
i) Snap-frozen pituitaries were thawed on ice. For nuclei isolation, a modified protocol from 52 was followed, all on ice. RNAse inhibitor (NEB MO314L) was added to the homogenization buffer (0.32 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 5mM CaCL2, 3mM Mg(Ac)2, 0.1% IGEPAL CA-630), 50% OptiPrep (Stock is 60% Media from Sigma; cat# D1556), 35% OptiPrep and 30% OptiPrep right before isolation. Each pituitary was homogenized with 10 strokes (loose pestle) in an individual autoclaved dounce glass homogenizer (1ml, VWR cat# 71000-514). The homogenate was filtered through a 40-micrometer cell strainer. The volume was measured and an equal volume of 50% OptiPrep was added. The gradient was centrifuged using an SW41 rotor at 9200 rpm and at 4C for 25 min. Nuclei were collected from the interphase between 30% and 35% OptiPrep. The nuclei were washed, resuspended either in 1x nuclei dilution buffer for snATACseq (10X Genomics) or in 1X PBS/0.04% BSA for snRNAseq, and counted on an Invitrogen Countess II cell counter. ii) Dissociated pituitary cells were cryopreserved in 90% FBS/10% DMSO and shipped to New York on dry-ice. Upon arrival, cells were stored in liquid nitrogen. On the day of an experiment, the vial was removed from liquid nitrogen on dry-ice, thawed quickly in a 37C waterbath, and cells were serially diluted with 10% FBS /DMEM until a minimum volume of 8ml was reached. Cells were centrifuged at 300xg for 5min, resuspended in 1ml of medium and counted. Nuclei were isolated from all cells remaining after the scGEM Drop-seq RNA-seq assay was done. Cells were centrifuged at 500g for 5min at 4C. The supernatant was carefully removed and 0.1X lysis buffer (1x: 10mM Tris-HCL pH 7.5, 10mM NaCl, 3mM MgCl2, nuclease-free H2O, 0.1% v/v NP-40, 0.1% v/v Tween-20, 0.01% v/v digitonin) was added. After 5min incubation on ice, 1ml of wash buffer was added. The nuclei were pelleted at 500xg for 5min at RT, resuspended either in 1X PBS/0.04% BSA for scGEM Drop-seq RNA-seq or in diluted nuclei buffer for snATAC-seq. Nuclei were counted and the concentration adjusted to run the given assay.
snATACseq was performed as described (10x Genomics, Pleasanton, CA), following the Chromium Single Cell ATAC Reagent Kits V1 User Guide. Nuclei were counted on a Countess instrument, and the final concentration was used to calculate the volume of nuclei stock to use. Transposition was performed in 10 microliter at 37C for 60min. Then the Chromium Chip E (PN-2000121) was loaded to target 4,000-10,000 nuclei. Barcoding was performed in the emulsion for 12 cycles following the Chromium protocol. Each library was tagged with a different index for multiplexing (Chromium i7 Sample Index N, Set A kit PN-3000262). Quality control and quantification of the libraries were assessed on a Bioanalyzer (High-Sensitivity DNA Bioanalyzer kit). Sequencing was carried out at the NYGC on Illumina Novaseq using 98+26 paired-end reads.

value: dissociated, cryopreserved pituitaries


title: cell transfected siFra-1-rep2

value: B


title: cell transfected siCTL-rep2


title: Female_Pit_2

value: Female


title: Female_Pit_1


title: non transfected cells-rep2

value: NT


title: cell transfected siFra-1-rep1


title: cell transfected siCTL-rep1


title: non transfected cells-rep3


title: FrozPit-FM3


title: V3_post

description: RRBS

value: CLL

value: Unmutated


title: RNA-seq in the CLIP deletion mESC, biological replciate 2

description:
mRNA
RNA-seq in the CLIP_deletion mESC, biological replicate 2, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv

value: the CLIP deletion mESCs

tag: genetic background


title: V3_pre

description: RRBS


title: 6h amp media rep3

city: Brookings

first: Nicholas

middle: C.

last: Butzin

state: SD

address: 1224 Medary Ave.

institute: South Dakota State University

data_processing:
Fastq files were aligned to NC_000913.3 and the plasmid p24KmNB82 using Geneious v. 11.0.3.
Raw read counts were calculated using 'calculate expression tool' in Geneious v. 11.0.3
Supplementary_files_format_and_content: Table with raw read counts for every gene and sample; comma separated values

molecule: polyA RNA

organism: Escherichia coli

source_name: 6h fresh media with ampicillin

growth_protocol: Cultures were grown in modified MMB+ media, which consists of the following: K2HPO4 (10.5 mg/ml), KH2PO4 (4.5 mg/ml), (NH4)2SO4 (2.0 mg/ml), C6H5Na3O7 (0.5 mg/ml), NaCl (1.0 mg/ml), 2 mM MgSO4 x 7H2O, 100 µM CaCl2, thiamine (10 µg/ml), 0.5% glycerol, amino acids (40 µg/ml) and kanamycin (25 µg/ml)

extract_protocol:
Samples were treated with Tween (0.2%) and chloramphenicol (5 µg/ml) then immediately filtered out of solution and resuspended in lysis buffer containing 25 mM Tris pH 8.0, 25 mM NH4Cl, 10 mM MgOAc, 0.8 % Triton X-100, 0.1 U/µL RNase-free DNase I, 1 U/μL. Then immediately frozen in liquid nitrogen and stored at -80 C.  Frozen pellets were thawed on ice and centrifuged for 20 minutes at 16,000 x g at 4 °C with 0.3% Sodium Deoxychorate to pellet particulate matter. The supernatant was then removed and RNA was column purified (Qiagen miRNeasy cat. No. 217004) before removing rRNA (Invitrogen RiboMinus cat no. K155004) Superase-In, ~60 U/µl ReadyLyse Lysozyme, 1.55 mM Chloramphenicol, and 17 μΜ 5′-guanylyl imidodiphosphate (GMPPNP).
cDNA libraries were prepared and multiplexed according to manufacturer's protocols: NEBNext Ultra II cat no. E7103S. Libraries were then pooled and sequenced by Illumina HiSeq paired-end sequencing.

treatment_protocol: Stationary phase cultures were diluted 1/100 into fresh MMB+ media and collected after three hours or six hours. Ampicillin treated cultures also contained 100 ug/ml ampicillin. IPTG treated cultures contained 100 uM IPTG.

tag: plasmid

value: ampicillin (6h)


title: RNA-seq in the CLIP deletion mESC, biological replciate 1

description:
mRNA
RNA-seq in the CLIP_deletion mESC, biological replicate 1, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv


title: FrozPit-FM2

value: 10-12 weeks

tag: source type


title: V2_post

description: RRBS

value: post-treatment


title: 6h amp media rep2


title: small RNA-seq in the Ago2 deletion mESC, biological replicate 1

description:
small RNA
small RNA-seq in the Ago2_deletion mESC, biological replicate 1, the microRNA expression level is quantified in the processed file "smallRNAseq_TPM.csv"
smallRNA_TPM.csv

value: Ago2 deletion mESCs


title: non transfected cells-rep1

tag: batch

value: T1- transcriptome 1

tag: replicate


description:
mRNA
RNA-seq in the wild-type mESC, biological replicate 2, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv

value: the wild-type mESCs


title: FrozPit-FM1


title: 6h amp media rep1


description:
mRNA
RNA-seq in the wild-type mESC, biological replicate 1, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv


title: FrozPit-MM3

value: Male


title: RNA-seq in the Ago2 deletion mESC, biological replicate 4

description:
mRNA
RNA-seq in the Ago2_deletion mESC, biological replicate 4, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv


title: FrozPit-MM2


title: 3h queueing amp rep3

source_name: 3h queueing amp

value: IPTG+ampicillin


title: V1_pre

description: RRBS

tag: patient id

value: pre-treatment


title: RNA-seq in the Ago2 deletion mESC, biological replicate 3

description:
mRNA
RNA-seq in the Ago2_deletion mESC, biological replicate 3, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv


title: MM_Pit_6

description: Male Mouse.  10Xv3 chemistry

data_processing:
SC RNA-seq data were processed using the Cell Ranger pipeline v3.0.2, and aligned to the mm10 transcriptome.
Genome_build: mm10
Supplementary_files_format_and_content: UMI count matrices in Hierarchical Data Format HDF5 for cell barcodes

growth_protocol: Animals were on a 12-hour on, 12-hour off light cycle, with lights on at 7 AM and off at 7 PM.

extract_protocol:
For the male samples, the following standard dissociation protocol was used: upon collection, pituitaries were digested with collagenase and dispersed with pancreatin, and the dissociated cells collected acutely. About 700,000-2.5x10^6 cells were collected from single pituitaries and placed in 90% FBS/10% DMSO in individual tubes for cryopreservation and shipped to New York on dry-ice. For the female samples, an improved protocol involving gentle dissociation was used as follows: a) Pituitary removal: Immediately following decapitation, remove the calvarium and gently lift up the cortex from the anterior end, then push it back to reveal the pituitary sitting in the sella turcica. With fine point forceps gently remove the diaphragma sellae covering the pituitary and remove the pituitary. Place it in warm Ca/Mg-free PBS containing 10% c/s FBS. b) Dispersion: Transfer the pituitary to a dish containing Ca/Mg-free PBS (no FBS) and remove the posterior lobe. Rinse 2x in PBS and to a 50 ml centrifuge tube containing a sterile flea-type stir bar and trypsin solution (final concentration at 0.37 mg/ml, Sigma T9935). Gently agitate at 37°C for 10 minutes. After gentle aspiration and expulsion steps, remove the supernatant, add MEM/10%ctFBS and put in an incubator at 37C while continuing the dissociation process. Repeat for 4 cycles, or until no more pieces are visible. Add 5 ml MEM/10%ctFBS and centrifuge. After resuspension, cells are counted and cryopreserved. Upon arrival in New York, all cryopreserved cell samples were stored in liquid nitrogen. On the day of an experiment, the vial was removed from liquid nitrogen on dry-ice, thawed quickly in a 37C waterbath, and cells were serially diluted with 10% FBS /DMEM until a minimum volume of 8ml. Cells were centrifuged at 300g for 5min, resuspended in 1ml of medium and counted.
GEM RNAseq was performed as described (10x Genomics, Pleasanton, CA; (24)), following the Single Cell 3’ Reagents Kits V2 or V3 User Guides. Cells were filtered, counted on a Countess instrument, and the final concentration was set at 1,000 cells/μl for cells. The amount of cells loaded on the chip was determined based on the 10X Genomics protocol. The 10X chip (Chromium Single Cell 3’ Chip kit A v2 PN-12036 or v3 chip kit B PN-2000060) was loaded to target 5,000-10,000 cells final. Reverse-transcription was performed in the emulsion and cDNA was amplified before library construction following the Chromium protocols v2 or v3. Each library was tagged with a different index for multiplexing (Chromium i7 Multiplex kit PN-12062). Quality control and quantification of the amplified cDNA were assessed on a Bioanalyzer (High-Sensitivity DNA Bioanalyzer kit). Sequencing was carried out at the NYGC on Illumina Novaseq using  98+26 paired-end reads.


title: RNA-seq in the Ago2 deletion mESC, biological replicate 2

description:
mRNA
RNA-seq in the Ago2_deletion mESC, biological replicate 2, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv


title: 3h queueing amp rep2


title: 3h queueing rep3

source_name: 3h queueing

value: IPTG


title: 3h queueing amp rep1


title: FrozPit-MM1

value: snap-frozen pituitaries


title: lcMBL3_normal

description: RRBS

tag: diagnosis

value: B cell


title: lcMBL3_CD19CD5

description: RRBS

value: low count MBL CD19+CD5+

tag: ighv


title: 3h queueing rep2


title: lcMBL2_normal

description: RRBS

tag: treatmentstatus


description:
mRNA
RNA-seq in the Ago2_deletion_and_CLIP_deletion mESC, biological replicate 4, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv

value: Ago2 deletion and CLIP deletion mESCs


title: 3h queueing rep1


title: lcMBL2_CD19CD5

description: RRBS


title: V2_pre

description: RRBS

value: CLL


title: lcMBL1_normal

description: RRBS


description:
mRNA
RNA-seq in the Ago2_deletion_and_CLIP_deletion mESC, biological replicate 3, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv


title: MM_Pit_4-1

description: Male Mouse.  10Xv3 chemistry

value: dissociated, cryopreserved pituitaries


title: MM_Pit_1v2

description: Male Mouse.  10Xv2 chemistry


title: V1_post

description: RRBS


description:
mRNA
RNA-seq in the Ago2_deletion_and_CLIP_deletion mESC, biological replicate 2, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv


title: FM_Pit_1-2

description: Female Mouse.  10Xv3 chemistry

value: Female


title: lcMBL1_CD19CD5

description: RRBS


description:
mRNA
RNA-seq in the Ago2_deletion_and_CLIP_deletion mESC, biological replicate 1, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv


title: HD3_naiBC

description: RRBS

value: naïve B cell CD5-


title: Trim71 CLIPseq in mESCs, size-matched Input control

description:
Size-matched Input Control
CLIP-seq on Trim71 Input sample size-matched control for Trim71-binding peak calling. the Trim71-binding peaks common in boht the biological replicate A and biological replicate B are reported in the file "Trim71_CLIPseq_Peaks_in_mESCs.csv"
Trim71_CLIPseq_peaks_in_mESCs.csv

value: FLAG-Trim71 mESCs

tag: antibody

value: FLAG-M2 (Sigma)


title: FM_Pit_1-1

description: Female Mouse.  10Xv3 chemistry

value: 10-12 weeks


title: RNA-seq in the Ago2 deletion mESC, biological replicate 1

description:
mRNA
RNA-seq in the Ago2_deletion mESC, biological replicate 1, the gene expression levels is quantified in the processed file "RNAseq_TPM.csv"
RNAseq_TPM.csv

value: Ago2 deletion mESCs


title: M-Pituitary

description: Male Mouse.  10Xv2 chemistry


title: HD3_memBC_CD5

description: RRBS

value: memory B cell CD5+


title: Trim71 CLIPseq in mESCs, Trim71 immunoprecipitation biological replicate B

description:
Trim71 bound mRNA
CLIP-seq on Trim71 Immunoprecipitation in the FLAG-Trim71 mESC, biological replicate B. the Trim71-binding peaks common in boht the biological replicate A and biological replicate B are reported in the file "Trim71_CLIPseq_Peaks_in_mESCs.csv"
Trim71_CLIPseq_peaks_in_mESCs.csv


title: MM_Pit_5

description: Male Mouse.  10Xv3 chemistry

value: Male


title: HD2_naiBC_CD5

description: RRBS

value: naïve B cell CD5+


title: Trim71 CLIPseq in mESCs, Trim71 immunoprecipitation biological replicate A

description:
Trim71 bound mRNA
CLIP-seq on Trim71 Immunoprecipitation in the FLAG-Trim71 mESC, biological replicate A, the Trim71-binding peaks common in boht the biological replicate A and biological replicate B are reported in the file "Trim71_CLIPseq_Peaks_in_mESCs.csv"
Trim71_CLIPseq_peaks_in_mESCs.csv


title: +CM AML CD34+, rep2

city: Duesseldorf

first: Thomas

last: Schroeder

address: Moorenstr. 5

department: Department of Hematology, Oncology and Clinical Immunology

institute: University of Duesseldorf, Medical Faculty

description:
CM_AML_CD34_sample11
CM_healthy_CD34_HSPC_VS_CM_AML_CD34_output_sorted.txt
CM_healthy_CD34_HSPC_VS_CM_AML_CD34_output_norm-matrix.txt

data_processing:
Cropping 12 bases from 5'-end using Trimmomatic (v.0.39)
2-pass alignment using STAR (v.2.7.2a): 1st pass: identification of of new splice junctions using all input data files together and addition to reference: 2nd pass: Alignment with combined reference for each input file individually.
Identification of raw counts using featureCounts (v.1.6.5)
Identification of normalized counts and differentially expressed genes using DEseq2 (v.1.24.0)
Calculation of FPKM-values using StringTie (v.2.0.3)
Genome_build: GRCh38.97
Supplementary_files_format_and_content: *.tab: tab-delimited txt-file:Stringtie-results TPM and FPKM values per gene
Supplementary_files_format_and_content: *_norm-matrix.txt: tab-delimited txt-file: DESeq2 normalized counts matrix
Supplementary_files_format_and_content: *_sorted.txt: tab-delimited txt-file: DEseq2 sorted list of differential expressed genes

source_name: CD34+ hematopoietic stem and progenitor cells (HSPC)

growth_protocol: Mononuclear cells (MNC) were obtained from BM aspirates following density gradient separation as previously described. CD34+ HSPC were positively selected from BM-derived MNC by immunomagnetic cell separation (Miltenyi Biotec, Bergisch-Gladbach, Germany). To investigate the influence of AML-derived CM on CD34+ HSPC functionality, 2x10^5/per well healthy CD34+ HSPC were cultivated on a 6-well plate in CM derived from AML cell lines (cytokines were then added to CM of cell lines for comparability) as well as from patient-derived MNC and CD34+ HSPC (CM from primary cells already contains cytokines from generation process). After 72 hours RNA was extracted.

extract_protocol:
RNA from hematopoietic cells (CD34+) was purified using the RNeasy Mini or Micro kit (both Qiagen, Hilden, Germany) according the manufacturer’s instructions.
DNase digested total RNA samples used for transcriptome analyses were quantified (Qubit RNA HS Assay, Thermo Fisher Scientific Inc., Waltham, MA, USA) and quality measured by capillary electrophoresis using the Fragment Analyzer and the ‘Total RNA Standard Sensitivity Assay’ or the Bioanalyzer assay “Eukaryote Total RNA Pico” (Agilent Technologies, Inc., Santa Clara, CA, USA). The mean RNA Quality Number (RQN) of the samples in this study was 8.9. The library preparation was performed according to the manufacturer’s protocol using the ‘NuGen Universal Plus mRNA-Seq Library Prep Kit’ for Illumina®. Briefly, 10 ng total RNA were used for poly(A) RNA selection, fragmentation, cDNA generation, adapter ligation, strand selection and library amplification. Bead purified libraries were normalized and finally sequenced on the HiSeq 3000/4000 system (Illumina Inc. San Diego, CA, USA) with a read setup of 1x150 bp. The bcl2fastq tool was used to convert the bcl files to fastq files as well for adapter trimming and demultiplexing.

treatment_protocol: To generate conditioned media (CM, supernatants) 2 × 10^6 cells of the AML cell lines HL-60, THP-1, and MV4-11 (all purchased from DMSZ, Braunschweig, Germany) were cultivated in T75 cm2 culture flasks for 3 days in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin/L-glutamine (PSG, all from Sigma-Aldrich Chemie GmbH, Taufkirchen Germany). For patient-derived CM 2 × 10^6/well BM-derived CD34+ AML cells and BM-derived CD34+ HSPC from healthy donors (HC) were cultivated on a 6-well plate under the same conditions. To properly maintain  CD34+ cells of patients with AML and healthy cells in culture for collection of CM, RPMI-1640 medium contained IL-3, IL-6, SCF and FLT3-ligand (all 10 ng/ml, PreproTech GmbH, Hamburg, Germany). Cells were incubated at 37°C, 5% CO2 under humidified conditions. Following incubation cell-free CM were subsequently harvested by filtration (Minisart® 0,45μm, Sartorius AG, Göttingen, Germany) and stored at -20°C for further experiments.

value: bone marrow derived CD34+ HSPC

tag: group

value: exposed to primary AML cell derived CM


title: +CM AML CD34+, rep3

description:
CM_AML_CD34_sample12
CM_healthy_CD34_HSPC_VS_CM_AML_CD34_output_sorted.txt
CM_healthy_CD34_HSPC_VS_CM_AML_CD34_output_norm-matrix.txt


title: HD2_naiBC

description: RRBS


title: HD2_memBC

description: RRBS

value: memory B cell CD5-


title: +CM THP-1, rep3

description:
CM_THP-1_sample9
CM_healthy_CD34_HSPC_VS_CM_THP-1_output_sorted.txt
CM_healthy_CD34_HSPC_VS_CM_THP-1_output_norm-matrix.txt

value: exposed to AML cell line THP-1 derived CM


title: +CM AML CD34+, rep1

description:
CM_AML_CD34_sample10
CM_healthy_CD34_HSPC_VS_CM_AML_CD34_output_sorted.txt
CM_healthy_CD34_HSPC_VS_CM_AML_CD34_output_norm-matrix.txt