title: ChIP_H3K4me3_Microglia_C57_Male_Adult_Basal_PND75_rep1

status: Public on Aug 18 2022

city: Québec

first: David

last: Gosselin

state: Quebec

address: CRCHUL - 2705 Boulevard Laurier, Bloc T - 2-34

department: Molecular Medicine

country: Canada

institute: Université Laval

data_processing:
Alignment: FASTQ files from sequencing experiments were mapped to the mouse mm10 reference genome. STAR (2.7.0f) with default parameters was used to map RNA-seq experiments. Bowtie2 (2.3.4.1) with default parameters was used to map ChIP-seq and ATAC-seq experiments. HOMER was used to convert aligned reads into “tag directories” for further analyses.
RNA-seq: For differential expression analyses, a table read count including relevant samples was first created using the analyzeRepeats.pl program of HOMER (v4.11; http://homer.ucsd.edu/homer/) with the following parameters: rna -noadj -condenseGenes -count exons -pc 3. Non-coding RNA and pseudogenes were then removed to retain only protein-coding mRNAs and the resulting dataset was used as input file for HOMER’s getDiffExpression.pl program, which leveraged R/DESeq2 for to assess differential expression. For data interpretation, only genes with 16 or more normalized reads were considered. Alternatively, Transcript Per Million-normalized datasets were generated using the analyzeRepeats.pl program of HOMER with the following parameters: rna -TPM -condenseGenes -count exons -pc 3. The base-2 logarithm of the TPM values was taken after adding a pseudocount of 1 TPM to each gene.
ChIP-seq: H3K4me3, H3K27ac ChIP-seq regions calling and differential analyses-For each condition, the repertoire of genomic regions positive for presence of H3K4me3 or H3K27ac were first generated using HOMER’s getDiffPeakReplicate.pl program, using all replicates and related input of a condition of interest. Parameters specified for H3K4me3 were as follow: -style histone. For H3K27ac, parameters were: -genome mm10 -region -size 500 -min Dist 1000 -L1. For differential enrichment assessments, relevant regions files were then first merged together with HOMER’s mergedPeaks.pl program with -size given parameter, and tag counts of each replicate annotated to each region using HOMER’s annotatePeak.pl program with mm10 -raw parameters used. For H3K27ac, data matrix was then used as input for HOMER’s getDiffExpression.pl program, which leveraged R/DESeq2 to assess differential enrichment. For read count tables output, each experiment was normalized to 10 million total reads (-simpleNorm default parameter specified in getDiffExpression.pl). For data interpretation, only regions with 16 or more normalized reads were considered.  Rpb2 ChIP-seq peak calling and analyses-Rpb2 peaks were first identified using HOMER’s getDiffPeakReplicate.pl program with the parameter -style factor specified and using two independent biological replicates of Rpb2 ChIP-seq experiments and associated inputs generated from Adult-Basal microglia. Rpb2 peaks from PND8-Ki67+ microglia (n=1) were identified using findPeaks.pl program with the -style factor parameter, using ChIP-seq and linked input data. To identify C1 and C2 promoter regions for which Rpb2 is present or absent, tables of genomic coordinates of C1 and C2 promoters were first generated by annotating C1 and C2 NCBI genes using HOMER’s annotatePeaks.pl program in tss mode using as input the list of NM RefSeq gene identifiers and specifying the following parameters: -list <relevant NM list.txt> -size -1000,500. Output was then curated to retain only gene regions included in the original NM list .txt file. Following this, C1 and C2 promoter regions were intersected with Rpb2 peaks files using BEDOPS (3.1.1 v2.4.39) with --ec --element-of-1 parameters specified.
ATAC-seq: ATAC-seq accessible chromatin region identification-Regions of accessible chromatin were called using HOMER’s findPeaks.pl program with the -style factor parameter.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Matrix tables with raw gene counts (Microglia_RNA_dUTP_rawdata.txt) or TPM-normalized values (Microglia_RNA_dUTP_TPMnormalization.txt) for every protein-coding genes gene and every RNA-seq samples.
Supplementary_files_format_and_content: Tab-delimited text files include peak coordinates (ChIP, ATAC) and enrichment over input (ChIP).

source_name: Microglia

extract_protocol:
Microglia isolation: Mice were first euthanized with an overdose of ketamine-xylazine administered by intra-peritoneal injection and subsequently perfused transcardially with PBS. Brain were then removed from the skull and immediately homogenized in a buffer solution (HBSS (Life Technologies, 14175-095), 1% BSA (Fisher), 1mM EDTA (Invitrogen), 1 mM sodium butyrate (Sigma B5887-1G), 1 uM Flavoperidol (Sigma F3055) by gentle mechanical dissociation on ice, using a 2 ml polytetrafluoroethylene pestle (Wheaton, 358026) as performed in Gosselin et al. 201417. Microglia were then enriched by Percoll (GE) gradient, washed and labeled with Live/Dead yellow dead stain (ThermoFisher), then incubated 1:100 with a CD16/CD32 receptor blocking antibody (1:50, eBioscience 14-0161, clone 93) for 20 min, and then incubated 1:100 with antibodies directed against cell surface proteins CD11b-BV421 (BioLegend 101251, clone M1/70), CD45-APC (BioLegend 103112, clone 30-F11), and CD44-APC-Cy7 (BioLegend 103028, clone IM7). For all ChIP-seq experiments, microglia were then further fixed with 1% PFA for 10 min at RT, permeabilized with reagents from ebioscience Foxp3 Transcription Factor staining buffer set (ThermoFisher 00-5523-00) and stained with anti-Ki67-Alexa 488 antibody (BD Pharmingen 561165, clone B56). Microglia were either analyzed with BD LSR or sorted with BD Aria II instruments (100 um nozzle). Flow cytometry results were analyzed using FlowJo software (Tree Star).
RNA-seq library preparation: Isolation and fragmentation of poly(A)   For RNA-seq, we generated 2 independent biological replicates per conditions (n=2). Following sorting, 200,000 microglia were pelleted and put into 100 ul lysis/Oligo d(T) Magnetic Beads binding buffer (100 mM Tris-HCl ph 7.5, 500 mM LiCl, 10 mM EDTA pH 8.0, 1% LiDS, 5 mM DTT, in water) and stored at -80C until processing. For human brain cortical RNA, 500 ng of RNA was diluted with proper volume of 2x lysis/ Oligo d(T) Magnetic Beads binding buffer to a final concentration of 1x lysis/Oligo d(T) Magnetic Beads binding buffer. For sequencing library preparation, samples were incubated with 20 ul Oligo d(T) Magnetic Beads (NEB, S1419S) in PCR strips for selection of polyadenylated (poly(A)) mRNA as follows: 2 min at 65 C on a PCR cycler and then 10 min at room temperature (RT). Samples were then placed on a collection magnet at RT, and washed serially with 180 ul of washing buffer 1 (RNA-WB1; 10 mM Tris-HCl pH 7.5, 0.15 M LiCl, 1 mM EDTA pH 8.0, 0.1% LiDS, 0.1% Triton X-100, in water) and then washing buffer 3 (RNA-WB3; 10 mM Tris-HCl, 0.15 M NaCl, 1 mM EDTA pH 8.0, in water). RNA was eluted from Oligo (t) beads by resuspending the beads in 50 ul of elution buffer (EB; 10 mM tris-HCl pH 7.5, 1 mM EDTA pH 8.0, in water) followed by incubation at 80C for 2 min on a PCR cycler. The samples were then placed back on the magnet, and the elution buffer supernatant containing the poly(A) RNA was carefully collected and placed on ice.   A second round of poly(A) RNA selection was then performed. Eluted Oligo d(T) beads were first washed on a magnet with 150 ul EB and then 150 ul 2X Oligo d(T) binding buffer (2x DTBB; 20 mM Tris-HCl pH 7.5, 1 M LiCl, 2 mM EDTA pH 8.0, 1% LiDS, 0.1% Triton X-100) and resuspended in 50 ul of 2x DTBB. Resuspended Oligo d(T) beads were then added to isolated RNA and incubated at 65 C and at RT as described above. Beads were then placed on a magnet and washed once with 150 ul of RNA-WB1 and then with 30 ul of 1x First-Strand buffer (made from 5x stock; Life Technologies) and transferred in a new PCR strip. Beads were collected on a magnet and resuspended in 10 ul of 2x SuperScript III buffer (Life Technologies, 1x First-Strand buffer, 10 mM DTT in water). Resuspended beads were then incubated at 94 C for 9 min to fragment poly(A) RNA. Beads were then placed back on a magnet, and the supernatant containing poly(A) RNA was carefully collected and placed on ice.  RNA-seq library preparation: Synthesis of cDNA  RNA in 2X SuperScript III buffer was incubated for 1 min at 50 C on a PCR cycler with 2.5 ul of the following mix: 1.5 ug Random Primer (Life Technologies 48190-011), 10 uM Oligo d(T) (Life Technologies 18418020), 10 units SUPERase-In (Ambion AM2696), 4 mM dNTP mix (Life Technologies 18427088), in water. Samples were then immediately placed on ice for 5 min. First-strand synthesis was then performed by incubation at 25 C for 10 min and 50 C for 50 min on a PCR cycler with 7.6 ul of the following mix: 0.2 ug actinomycin (Sigma A1410), 13.15 mM DTT (Life Technologies), 0.026% Tween-20, 100 units SuperScript III (Life Technologies kit 18080-044), in water. After incubation, RNA/DNA complexes were isolated by adding 36 ul of Agencourt RNAClean XP beads (Beckman Coulter A63987) and incubated for 10 min at RT and then 10 min on ice. Samples were then placed on a magnet and beads were washed twice with 150 ul of 75% EtOH. Following washings, beads were air-dried for 10-12 min and eluted with 10 ul of water.  Second-strand synthesis was then performed. RNA/DNA samples in 10 ul of water were incubated for 2.5 hours at 16 C with 5 ul of the following mix: 3x Blue Buffer (Enzymatics), 1.0 ul PCR mix (Affymetrix 77330), 2.0 mM dUTP (Affymetrix 77206), 1 unit RNAseH (Ezymatics Y9220L), 10 units DNA Polymerase I (Enzymatics P7050L), 0.03% Tween-20, in water). DNA was then purified by addition of 1.5 ul Sera-Mag SpeedBeads (Thermo Scientific, 651520505025), resuspended in 30 ul 20% PEG 8000/2.5 M NaCl, incubated at RT for 15 min and placed on a magnet for two rounds of bead washing with 80% EtOH. Beads were then air dried for 10-12 min and DNA was eluted from the beads by adding 40 ul of water. Supernatant was then collected on a magnet and placed on ice or stored at -20C until DNA blunting, poly(A)-tailing, and adapter ligation (see below).   RNA-seq final library preparation  Sequencing libraries were prepared from generated cDNA (RNA) by blunting, A-tailing, and adapter ligation as previously described using barcoded adapters (NextFlex, Bioo Scientific). Prior to final PCR amplification, RNA-seq libraries were digested by 30 min of incubation at 37C with Uracil DNA Glycosylase (final concentration of 0.134 units per ul of library volume; UDG, Enzymatics G5010L) to generate strand-specific libraries. Libraries were PCR-amplified for 12-15 cycles and size selected for fragments (200-500 for RNA-seq) by gel extraction (10% TBE gels, Life Technologies EC62752BOX). RNA-seq libraries were single-end sequenced for 76 cycles on an Illumina HiSeq 4000 (Illumina, San Diego, CA) according to manufacturer’s instruction.
Chromatin immunoprecipitation: Chromatin immunoprecipitation for histone modification H3K4me3, H3K27ac and Rpb2 and was performed as follows, using ~500,000 microglia per assay and n=2 independent biological replicates per conditions. First microglia were briefly thawed on ice and lysed by incubation in 1 ml of lysis buffer (0.5% IGEPAL CA-630, 10 mM HEPES pH 7.9, 85 mM KCl, 1 mM EDTA pH 8.0, in water) for 10 min on ice. Lysates were centrifuged at 800 RCF for 5 min at 4 °C, and pellets were resuspended in 200 ul of sonication/ immunoprecipitation buffer (10 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.5 mM EGTA, 0.1% Deoxcycholate, 0.5% Sarkosyl, in water). Sonication was performed with a Bioruptor Standard Sonicator (Diagenode) and consisted of two rounds of 15 min each, alternating stages of 30 sec “sonication-on” with 60 sec “sonication-off”. Twenty-two microliters of 10% Triton-X were then added to samples (1% final concentration) on ice, and lysates were cleared by centrifugation for 5 min at 18,000 RCF at 4 °C. Two microliters of supernatant were then set aside for input library sequencing controls. Supernatants were then immunoprecipitated on a rotator for two hours at 4 °C with antibody pre-bound to 17 ul Protein A Dynabeads (Life Technologies 10001D). The following antibodies were used: H3K4me3 (Millipore

value: WT

value: Ki67-Negative microglia

value: Male

tag: age at sacrifice (in days)

value: H3K4me3 (Millipore, 04-745)


title: mpMG_APOE44_2

status: Public on Jun 23 2022

type: SRA

city: Boston

first: Julia

last: TCW

state: Massachusetts

address: 700 Albany St.

country: USA

institute: Boston University

library_source: transcriptomic

data_processing:
Paired end reads were trimmed to 100bp reads for Illumina TruSeq adapters and matching read depth of other datasets
Reads were aligned to the hg38 genome, and samples were counted for reads, both using the STAR aligner (v2.5.3a).
Genes were filtered by expression abundance (Counts per million (CPM) >0.1 in 5 or more samples)
Library sizes were estimated by the trimmed mean of M-values normalization (TMM) using edgeR (v3.26.8)
Differential gene expression was predicted by linear mixed model analysis using the variancePartition package (v1.14.1) and DESeq2 (v.3.14)
Genome_build: hg38
Supplementary_files_format_and_content: mouseApoe_featureCounts.txt (Tab-delimited text file of raw read counts)

molecule: total RNA

organism: Mus musculus

source_name: APOE44 mouse primary microglia

growth_protocol: Primary microglia and astrocytes from mouse APOE 33, APOE 44, Apoe WT and Apoe KO cells were purified and cultured in human microglia and astrocyte media.

extract_protocol:
Total RNA was extracted from differentiated CNS cells in a single batch using the Total RNA isolation kit (Rneasy kit from Qiagen).
Libraries were prepared using NEBNext Ultra RNA Library Prep Kit for Illumina, and sequenced as paired-end 150bp reads on the Illumina HiSeq 4000.

treatment_protocol: No treatment

tag: cell type

value: Microglia

tag: apoe genotype

tag: batch

tag: Sex

value: F

tag: rin


title: ChIP_H3K4me3_Microglia_C57_Male_Adult_Basal_PND75_rep2

library_source: genomic

molecule: genomic DNA

tag: strain

tag: genotype

tag: treatment

tag: chip antibody


title: mpMG_APOE44_4


title: mpMG_APOE44_3

value: M


title: mpMG_APOE33_6

source_name: APOE33 mouse primary microglia


title: mpMG_APOE33_4


title: mpMG_APOE33_5


title: mpAst_APOEKO_4

source_name: APOEKO mouse primary astrocytes

value: Astrocytes

value: KO


title: mpAst_APOEKO_3


title: mpMG_APOE33_1


title: mpMG_APOE33_2


title: mpAst_APOEKO_2


title: mpAst_APOEKO_1


title: mpAst_APOE44_5

source_name: APOE44 mouse primary astrocytes


title: mpMG_APOEKO_3

source_name: APOEKO mouse primary microglia


title: mpMG_APOEKO_2


title: mpAst_APOE44_4


title: mpAst_APOE44_2


title: mpAst_APOE44_1


title: mpMG_APOE44_1


title: mpAst_APOE33_5

source_name: APOE33 mouse primary astrocytes


title: mpAst_APOE33_3


title: mpAst_APOE33_1


title: mpMG_ApoeWT_6

source_name: ApoeWT mouse primary microglia


title: mpMG_ApoeWT_3


title: mpMG_ApoeWT_4


title: mpAst_APOE33_2


title: mpMG_APOE33_3


title: mpAst_APOE33_4


title: mpMG_ApoeWT_1


title: mpMG_ApoeWT_2


title: mpAst_APOE33_6


title: mpAst_ApoeWT_5

source_name: ApoeWT mouse primary astrocytes


title: mpMG_ApoeWT_5


title: mpAst_ApoeWT_6


title: mpAst_APOE44_6


title: mpAst_ApoeWT_4


title: mpAst_ApoeWT_1


title: mpAst_ApoeWT_3


title: mpAst_ApoeWT_2


title: mpAst_APOE44_3


title: mmNUP98-NSD1_MSK_ChIP_MLL1_VTP_200nM_4d_REP1_200807_CGATGT_S2

status: Public on Jun 01 2022

first: Scott

last: Armstrong

state: MA

address: 450 Brookline Ave

department: pediatric oncology

institute: Dana Farber Cancer Institute

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the mouse (Gencode M24/mm10) genome using STAR (v2.7.5a; params --alignIntronMax 1 --alignEndsType EndToEnd --alignM  atesGapMax 2000 for ChIPseq analysis), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
ChIP-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75) and ngs.plot (pileup heatmaps). ChIPseq peaks were called using MACS2 with appropriate input samples used as controls.
TSS- and other interval-associated signals were assessed using bedtools coverage (v2.28.0).
Genome_build: Gencode M24/mm10
Supplementary_files_format_and_content: MACS2 peak output (BED format)

source_name: mouse leukemia

growth_protocol: For in vitro experiments using mouse leukemia cells, cells were maintained in in IMDM supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin (Gibco), 10 ng/mL mouse IL-3, 10 ng/mL mouse IL-6, and 10 ng/mL mouse SCF (Stem Cell Technologies). All cells were maintained at 37C and 5% CO2.

extract_protocol:
The cells were collected and washed twice in ice-cold PBS followed by crosslinking in 1% formaldehide (methanol free) for 7-8 minutes. Excess of formaldehyde was quenched by addition of Tris pH 8.0 to 100nM final and Glycine 125 mM final. Chromatin was sheared using Covaris E220 for 30 min with power 140; duty 5; bursts 200/sec. We then used a 5ul aliquot of sherared chromatin to decrosslink in order to test completeness of shearing. Chromatin passed QC if fragments between 100-700bp were >90%. For immunoprecipitation we used chromatin from 20x10^6 cells, 5ug of specific antibody and Protein-A/G magnetic beads (Dynal). Following roatation at 4 degrees overnight, the immune complexes were wased in Mixed Micelle Buffer, buffer 500, LiCl/detergent solution and TE buffers. DNA was eluted from the beads using 100mM NaHCO3 100mM NaCl 1% SDS and purified using AmpureXP beads.
1-10 ng of ChIP'ed DNA was used for the library preparation using the SMARTer ThruPLEX DNA-Seq Kit (Takara)
ChIP-Seq, Sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) with 37bp, paired-end reads.

treatment_protocol: For in vitro experiments using mouse leukemia cells, cells were treated with DMSO or 2 micromolar VTP50469 for the specified length of time.

tag: cell line

tag: antibody

tag: antibody manufacturer

value: Bethyl

tag: antibody catalog number

tag: agent

tag: timepoint

value: 96 hours


title: mmNUP98-NSD1_MSK_ChIP_input_VTP_200nM_4d_REP1_200807_ACAGTG_S5


title: mmNHD13_ChIP_MEN1_VTP_2uM_96h_REP1_210716_TGACCA_S4


title: mmNHD13_ChIP_MEN1_DMSO_96h_REP1_210716_TTAGGC_S3

value: DMSO


title: mm202C-H9C_ChIP_MLL1_VTP_2uM_REP1_201029_GCCAAT_S6

value: 72 hours


title: mmNUP98-NSD1_MSK_ChIP_MLL1_DMSO_4d_REP1_200807_ATCACG_S1


title: mmNHD13_ChIP_input_DMSO_96h_REP1_210716_CAGATC_S7


title: mmNHD13_ChIP_input_VTP_2uM_96h_REP1_210716_ACTTGA_S8


title: mm202C_ChIP_MEN1_DMSO_96h_REP1_210716_ATCACG_S1


title: mm202C-H9C_ChIP_MLL1_DMSO_72h_REP1_201029_ACAGTG_S5


title: mm202C-H9C_ChIP_input_DMSO_72h_REP1_201029_ATGTCA_S15


title: mm202C_ChIP_H3K4me3_VTP_2uM_96h_REP1_210716_CTTGTA_S12

value: Abcam


title: mmNUP98-NSD1_MSK_RNA_VTP_200nM_72h_REP1_200618_CGTACG_S10

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human (Gencode GRCh38/hg38) or mouse (Gencode M24/mm10) genome using STAR (v2.7.5a, sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
RNA-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75).
Raw per-gene counts calculated with HTSeq (htseq-count, v0.6.1pl).
Differential RNA-seq expression was calculated using the BioConductor DESeq2 package (v1.24,0), using raw unnormalized per-gene counts from deduplicated BAMs.
Genome_build: GRCh38/hg38 and M24/mm10
Supplementary_files_format_and_content: HTSeq per-gene read counts (TXT)

growth_protocol: For in vitro experiments using mouse leukemia cells, cells were maintained in in IMDM supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin (Gibco), 10 ng/mL mouse IL-3, 10 ng/mL mouse IL-6, and 10 ng/mL mouse SCF (Stem Cell Technologies). All cells were maintained at 37C and 5% CO2. For human AML patient derived xenograft (PDX) experiments, mice were transplanted with 2-5x10^5 nucleated cells.

extract_protocol:
For RNA isolation from cell lines, 1x10^6 cells were lysed in 350 µl of RLT buffer and RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturers instructions for cell ine experiments.
Poly-A tail selection and library preparation were performed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA).
Sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) with 37bp, paired-end reads.

treatment_protocol: For in vitro experiments using mouse leukemia cells, cells were treated with DMSO or 2 micromolar VTP50469 for the specified length of time. For in vivo AML PDX experiments, mice were treated fed control chow or chow containing 0.1% VTP50469.

value: NUP98-NSD1 mouse leukemia


title: mm202C_ChIP_MEN1_VTP_2uM_96h_REP1_210716_CGATGT_S2


title: mm202C_ChIP_input_VTP_2uM_96h_REP1_210716_GTGAAA_S18


title: mmNUP98-NSD1_MSK_RNA_DMSO_72h_REP1_200618_GTGAAA_S7


title: mmNUP98-NSD1_MSK_RNA_DMSO_72h_REP2_200618_GTGGCC_S8


title: mm202C_ChIP_H3K4me3_DMSO_96h_REP1_210716_GGCTAC_S11


title: mmNUP98-NSD1_MSK_RNA_VTP_200nM_72h_REP3_200618_GGTAGC_S12


title: mmNHD13_RNA_VTP_2uM_96h_REP3_210302_TACAGC_S12

source_name: NUP98-HOXD13 mouse leukemia

value: NUP98-HOXD13 mouse leukemia


title: mm202C_ChIP_input_VTP_2uM_96h_REP1_210716_GCCAAT_S6


title: mmNUP98-NSD1_MSK_RNA_VTP_200nM_72h_REP2_200618_GAGTGG_S11


title: mmNHD13_RNA_VTP_2uM_96h_REP2_210302_TAATCG_S11


title: mmNUP98-NSD1_MSK_RNA_DMSO_72h_REP3_200618_GTTTCG_S9


title: mmNHD13_RNA_VTP_2uM_96h_REP1_210302_GACGAC_S10


title: mmNHD13_RNA_VTP_2uM_72h_REP2_210302_CCAACA_S5


title: mmNHD13_RNA_VTP_2uM_72h_REP1_210302_CATTTT_S4


title: mmNHD13_RNA_VTP_2uM_72h_REP3_210302_CGGAAT_S6


title: mmNHD13_RNA_DMSO_96h_REP2_210302_CTATAC_S8


title: mmNHD13_RNA_DMSO_96h_REP1_210302_CTAGCT_S7


title: mmNHD13_RNA_DMSO_72h_REP3_210302_CATGGC_S3


title: mmNHD13_RNA_DMSO_72h_REP2_210302_CAGGCG_S2


title: mm202C_RNA_VTP_2uM_6d_REP3_210419_ATTCCT_S12

source_name: NUP98-HOXA9 mouse leukemia

value: NUP98-HOXA9 mouse leukemia

value: 6 days


title: mm202C_RNA_VTP_2uM_6d_REP2_210419_ATGAGC_S11


title: mm202C_RNA_DMSO_6d_REP3_210419_GGTAGC_S9


title: mmNHD13_RNA_DMSO_72h_REP1_210302_CACTCA_S1


title: mm202C_RNA_DMSO_6d_REP2_210419_GAGTGG_S8


title: mm202C_RNA_DMSO_6d_REP1_210419_CGTACG_S7


title: mm202C-H9C_RNA_VTP_2uM_72h_REP2_200914_CATTTT_S11


title: mm202C-H9C_RNA_VTP_2uM_72h_REP3_200914_CCAACA_S12


title: mm202C-H9C_RNA_VTP_2uM_72h_REP1_200914_CATGGC_S10


title: mm202C-H9C_RNA_DMSO_72h_REP2_200914_CACTCA_S8


title: mm202A_RNA_VTP_2uM_6d_REP1_210419_GTGAAA_S4

source_name: NUP98-JARID1A mouse leukemia

value: NUP98-JARID1A mouse leukemia


title: mm202A_RNA_VTP_2uM_6d_REP3_210419_GTTTCG_S6


title: mm202C-H9C_RNA_DMSO_72h_REP3_200914_CAGGCG_S9


title: mm202A_RNA_DMSO_6d_REP1_210419_CCAACA_S1


title: mm202C-H9C_RNA_DMSO_72h_REP1_200914_CACGAT_S7


title: mm202A_RNA_DMSO_6d_REP3_210419_GTCCGC_S3


title: mm202A-J1AC_RNA_VTP_2uM_72h_REP2_200914_TCGAAG_S23


title: mm202A-J1AC_RNA_VTP_2uM_72h_REP3_200914_TCGGCA_S24


title: mm202A-J1AC_RNA_VTP_2uM_72h_REP1_200914_TCCCGA_S22


title: mm202A-J1AC_RNA_DMSO_72h_REP3_200914_TCATTC_S21


title: mm202A-J1AC_RNA_DMSO_72h_REP1_200914_TACAGC_S19


title: mmNHD13_ChIP_MLL1_DMSO_96h_REP1_210420_TTCTGTGT_S1

extract_protocol:
The cells were collected and washed twice in ice-cold PBS followed by crosslinking in 1% formaldehide (methanol free) for 7-8 minutes. Excess of formaldehyde was quenched by addition of Tris pH 8.0 to 100nM final and Glycine 125 mM final. Chromatin was sheared using Covaris E220 for 30 min with power 140; duty 5; bursts 200/sec. We then used a 5ul aliquot of sherared chromatin to decrosslink in order to test completeness of shearing. Chromatin passed QC if fragments between 100-700bp were >90%. For immunoprecipitation we used chromatin from 20x10^6 cells, 5ug of specific antibody and Protein-A/G magnetic beads (Dynal). Following roatation at 4 degrees overnight, the immune complexes were wased in Mixed Micelle Buffer, buffer 500, LiCl/detergent solution and TE buffers. DNA was eluted from the beads using 100mM NaHCO3 100mM NaCl 1% SDS and purified using AmpureXP beads.
1-10 ng of ChIP'ed DNA was used for the library preparation using the SMARTer ThruPLEX DNA-Seq Kit (Takara)
Sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) with 37bp, paired-end reads.

value: Bethyl A300-086A


title: mmNHD13_ChIP_MLL1_VTP_2uM_96h_REP1_210420_TCTGCTGT_S2


title: mm202A-J1AC_RNA_DMSO_72h_REP2_200914_TATAAT_S20


title: mm202C_RNA_VTP_2uM_6d_REP1_210419_ACTGAT_S10


title: mmNHD13_ChIP_input_VTP_2uM_96h_REP1_210420_ATTCCT_S6


title: mmNHD13_ChIP_input_DMSO_96h_REP1_210420_ACTGAT_S5


title: mm202A-J1AC_ChIP_MLL1_DMSO_96h_REP1_210322_TTAGGC_S3


title: mm202A-J1AC_ChIP_MLL1_VTP_2uM_96h_REP1_210322_TGACCA_S4


title: mm202A-J1AC_ChIP_input_VTP_2uM_96h_REP1_210322_CGATGT_S2


title: mm202A-J1AC_ChIP_input_DMSO_96h_REP1_210322_ATCACG_S1


title: mmNHD13_RNA_DMSO_96h_REP3_210302_GTGATC_S9


title: mm202A_RNA_DMSO_6d_REP2_210419_GTAGAG_S2


title: mmNHD13_ChBID_input_DMSO_96h_REP1_210120_GTGAAA_S6

data_processing:
Library strategy: BioChIP-seq
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the mouse (Gencode M24/mm10) genome using STAR (v2.7.5a; params --alignIntronMax 1 --alignEndsType EndToEnd --alignM  atesGapMax 2000 for BioChIPseq analysis), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
ChIP-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75) and ngs.plot (pileup heatmaps). ChIPseq peaks were called using MACS2 with appropriate input samples used as controls.
TSS- and other interval-associated signals were assessed using bedtools coverage (v2.28.0).
Genome_build: Gencode M24/mm10
Supplementary_files_format_and_content: MACS2 peak output (BED format)

extract_protocol:
The cells were collected and washed twice in ice-cold PBS followed by crosslinking in 1% formaldehide (methanol free) for 7-8 minutes. Excess of formaldehyde was quenched by addition of Tris pH 8.0 to 100nM final and Glycine 125 mM final. Chromatin was sheared using Covaris E220 for 30 min with power 140; duty 5; bursts 200/sec. We then used a 5ul aliquot of sherared chromatin to decrosslink in order to test completeness of shearing. Chromatin passed QC if fragments between 100-700bp were >90%. For immunoprecipitation we used chromatin from 20x10^6 cells, and immunoprecipitated biotinylated fusion proteins using streptavidin coated magnetic  Dynabeads M-280 Streptavidin (Thermo Fisher 11205D). Precipitates were washed sequentially at room temperature with 2% SDS, followed by ice cold low salt wash (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris-HCl, pH8.1, 150mM NaCl), high salt wash (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM TrisHCl, pH 8.1, 500mM NaCl), LiCl wash (0.25M LiCl, 1% IGEPAL CA-630, 1% deoxycholic acid, 1mM EDTA, 10mM Tris-HCl, pH 8.1) and TE wash (1mM EDTA, 10mM Tris-HCl, pH 8.1) and eluted in elution buffer (1% SDS, 0.1 M NaHCO3). Immunoprecipitated DNA fragments were eluted and de-crosslinked in 100 mM NaHCO3, 100 mM NaCl, 1% SDS, then purified using Agencourt AMPure XP beads (VWR 75803-122) at a ratio of 1:1.2 in preparation for generation of Illumina-compatible sequencing libraries.
1-10 ng of ChIP'ed DNA was used for the library preparation using the SMARTer ThruPLEX DNA-Seq Kit (Takara)
Sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) with 37bp, paired-end reads.

tag: streptavidin-coated beads

value: Dynabeads M-280 Streptavidin

value: ThermoFisher

tag: biochip target

value: biotinylated NUP98-HOXD13


title: mT5_NTPL511_RNA_VTP_chow_14d_REP2_210419_ACTTGA_S8

organism: Homo sapiens

source_name: NUP98-NSD1 human AML PDX

tag: cell type

value: NUP98-NSD1 human AML PDX

value: 14 days

value: 0.1percent VTP50469 chow


title: mmNHD13_ChBID_NUP98-HOXD13_DMSO_96h_REP1_210120_ATGTCA_S3


title: mmNHD13_ChBID_NUP98-HOXD13_VTP_2uM_96h_REP1_210120_CCGTCC_S4


title: mT11_NTPL511_RNA_VTP_chow_14d_REP5_210419_AGTTCC_S14


title: mT12_NTPL511_RNA_VTP_chow_14d_REP6_210419_ATGTCA_S15


title: CPCT0021_RNA_VTP_chow_36d_REP3_210430_AGTTCC_S14

source_name: NUP98-JARID1A human AML PDX

value: NUP98-JARID1A human AML PDX

value: 36 days


title: mC6_NTPL511_RNA_control_14d_REP3_210419_CTTGTA_S12

value: untreated


title: mC4_NTPL511_RNA_control_14d_REP1_210419_TAGCTT_S10


title: CPCT0021_RNA_VTP_chow_36d_REP1_210430_CTTGTA_S12


title: mT10_NTPL511_RNA_VTP_chow_14d_REP4_210419_AGTCAA_S13


title: CPCT0021_RNA_VTP_chow_36d_REP2_210430_AGTCAA_S13


title: CPCT0021_RNA_control_36d_REP1_210430_GATCAG_S9


title: mm202A_RNA_VTP_2uM_6d_REP2_210419_GTGGCC_S5


title: CPCT0021_RNA_control_36d_REP2_210430_TAGCTT_S10


title: Hi-C_TNBC_Tissue1

status: Public on May 06 2022

city: Daejeon

first: Taemook

last: Kim

address: 291 Daehak-ro, Yuseong-gu

department: Biological sciences

country: South Korea

institute: Korea Advanced Institute of Science and Technology

data_processing:
(Hi-C) Hi-C data were analyzed according to the HiC-Pro protocol.
Raw paired-end data were separately aligned to the human reference genome (hg19) and filter out artifacts (self-ligation, singleton, dangling end, and dumped pairs).
The extracted valid-paired reads were used to build a contact matrix for each resolution. (10, 20, 40, 100 and 500kb) and to normalize with iterative correction and eigenvector (ICE) method.
The GENOVA was used to draw the figure such as contact map, relative contact probability (RCP), cis-interaction within each chromosome, and trans-interaction between chromosomes.
The scatter plot, density plot, Venn diagram, and boxplot associated with a contact at peaks were drawn using an R package.
Genome_build: hg19
Supplementary_files_format_and_content: hic

source_name: Triple negative breast cancer tissue, patient1

growth_protocol:
All cell lines, which is a normal cell and breast cancer cell, used in this study were purchased from ATCC. Human mammary epithelial cell (HMEC, PCS-600-010, ATCC) was cultured in mammary epithelial cell basal medium (PCS-600-030, ATCC) supplemented with mammary epithelial cell growth kit (PCS-600-040) as ATCC suggested. T47D (HTB-133) was cultured in RPMI-1640 (ATCC, 30-2001) supplemented with 10% FBS (ATCC, 30-2020) and 0.2 units/ml of human insulin (Life Technologies, 12585-014). BT549 (HTB-122) was cultured in RPMI-1640 supplemented with 10% FBS and 0.023 units/ml of human insulin. HCC1954, HCC70, and ZR-75-30 were cultured in RPMI-1640 supplemented with only 10% FBS. All cell lines were grown in the humidified incubator at 37°C containing 5% CO2.
The frozen fresh tumor samples were collected from resected breast tumors from patients at the Seoul National University Hospital (SNUH) in South Korea with informed consent and institutional IRB approval. The frozen tissues provided are as follows: Contralateral breast normal tissue and triple negative breast cancer tissue. These frozen samples were snap-frozen in liquid nitrogen and stored at -80°C.

extract_protocol:
(Hi-C) The in situ Hi-C experiments for cells and tissues were performed according to the Arima-HiC protocol (Arima Genomics, Inc.; Cat #A160259).
(Hi-C) The Hi-C library was generated using the Arima-HiC kit (Arima Genomics, Inc.; Cat #A510008)

tag: tissue/cell type

tag: histological subtype

value: Triple negative breast cancer IDC


title: Hi-C_TNBC_Tissue3

source_name: Triple negative breast cancer tissue, patient3

extract_protocol:
(Hi-C) The in situ Hi-C experiments for cells and tissues was performed according to the Arima-HiC protocol (Arima Genomics, Inc.; Cat #A160259).
(Hi-C) The Hi-C library was generated using the Arima-HiC kit (Arima Genomics, Inc.; Cat #A510008)


title: Hi-C_BT549_rep1

source_name: Triple negative breast cancer B cell

value: Triple negative breast cancer B


title: Hi-C_HCC70_rep1

source_name: Triple negative breast cancer A cell

value: Triple negative breast cancer A


title: CPCT0021_RNA_control_36d_REP3_210430_GGCTAC_S11


title: Hi-C_HCC1954_rep1

source_name: Her2+ cell

value: Her2 positive


title: Hi-C_T47D_rep1

source_name: Luminal A cell

value: Luminal A


title: mT4_NTPL511_RNA_VTP_chow_14d_REP1_210419_CAGATC_S7


title: mT6_NTPL511_RNA_VTP_chow_14d_REP3_210419_GATCAG_S9


title: Hi-C_ZR7530_rep1

source_name: Luminal B cell

value: Luminal B


title: D91_M

status: Public on May 01 2022

city: Oxford

first: Adam

last: Cribbs

address: Windmill Road, Oxford, OX3 7LD, UK

country: United Kingdom

institute: University of Oxford

data_processing:
The fastq files were pseudo-aligned by Kallisto with the human genome hg38. The samples with the alignment rate < 60% were excluded from the downstream analysis.
The count matrix was generated by Kallisto and analyzed for differential expression by DESeq2 v1.35.0
The coun matrix was further normalized by DESeq2 and used in the multi-omic factor analysis, MOFA2.
Assembly: Human genome hg38
Supplementary files format and content: count matrix generated by Kallisto

source_name: Uterus

extract_protocol:
Tissues were first cryomilled. Total RNA was harvested with Trizol and then purified with Direct-zol RNA Miniprep (R2052).
The total RNA was poly(A)-selected with NEBNext Poly(A) mRNA magnetic isolation module (NEB #E7490) and the library was constructed with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina

tag: patient

value: Uterine fibroid patient

tag: tissue

value: Myometrium

value: mut

value: HMB

value: Secretory

tag: batch


title: mC5_NTPL511_RNA_control_14d_REP2_210419_GGCTAC_S11

value: NUP98-NSD1 human AML PDX


title: D78_M

value: WT

value: Proliferative


title: D71_M


title: D84_M

value: No

value: Inactive


title: D65_M

value: Unknown


title: D59_M


title: D67_M


title: D64_M


title: Hi-C_HMEC_rep1

source_name: human mammary epithelial cell

value: Primary Mammary Epithelial Cell


title: D47_M

value: Non-uterine fibroid patient


title: D48_M


title: D44_M


title: D45_M


title: D43_M


title: D42_M


title: Hi-C_Normal_Tissue1

source_name: Contralateral breast tissue, patient N1

value: Contralateral breast tissue, patient N1


title: D38_M


title: D37_M


title: D39_M


title: D31_M


title: D35_M


title: Hi-C_TNBC_Tissue2

source_name: Triple negative breast cancer tissue, patient2

value: Triple negative breast cancer IDC


title: D30_M


title: D28_M


title: D29_M


title: D26_M


title: D23_M


title: D19_M


title: D24_M


title: D18_M


title: D20_M


title: D79_M


title: D21_M


title: D16_M


title: D119_M


title: D120_M


title: D108_M


title: D111_M


title: D96_F

value: Fibroid


title: D91_F


title: D87_F


title: D85_F


title: D88_F


title: D90_F


title: D84_F


title: D83_F


title: D77_F


title: D72_F


title: D17_M


title: D126_M


title: D65_F


title: D59_F


title: D123_M


title: D64_F


title: D57_F


title: D116_M


title: D54_F


title: D125_M


title: D71_F


title: D5_F


title: D53_F


title: D67_F


title: D52_F


title: D4_F


title: D62_F


title: D48_F


title: D80_F


title: D30_F


title: D46_F


title: D42_F


title: D34_F


title: D39_F


title: D31_F


title: D38_F


title: D29_F


title: D26_FC


title: D28_F


title: D24_F


title: D23_F


title: D20_F


title: D2_F


title: D19_F


title: D123_F


title: D121_F


title: D18_F


title: D126_F


title: D120_F


title: D125_F


title: D119_F


title: D100_F


title: D113_F


title: D109_F


title: D10_F


title: D88_E

value: Endometrium


title: D91_E

value: Uterine fibroid patient


title: D84_E


title: D64_ED


title: D79_E


title: D61_LE


title: D61_ED


title: D45_E


title: D59_DE


title: D43_E


title: D59_LE


title: D38_E


title: D40_E


title: D42_E


title: D35_ED


title: D32_E


title: D30_EL


title: D30_ED


title: D31_DE


title: D24_E


title: D29_EL


title: D28_E


title: D23_E


title: D21_E


title: D20_E


title: D137_E


title: D16_E


title: D29_ED


title: D130_E


title: D17_E


title: D91_C

value: Pseudocapsule


title: D108_E


title: D67_C


title: D61_C

value: mut


title: D39_C


title: D31_C


title: D42_C


title: D28_C

value: HMB


title: D23_C


title: D20_C

value: Secretory


title: D18_C


title: D126_C


title: D74_C


title: MV411_RNA_VTP_50nM_3d_REP1_191024_CTAGCT_S10

status: Public on Mar 10 2022

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human genome (GRCh37/hg19 Gencode r19) using STAR (v2.7.5a), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4). Final “deduped” .BAM files were indexed using SAMtools (v1.95).
For RNA-seq, raw counts calculated with HTSeq (htseq-count, v0.11.2). Duplicate reads were marked and removed with picard tools (v2.9.4).
Raw counts were used to calculate differential expression values with the R Bioconductor DESeq2 package (v1.24.0). Results were filtered to exclude non-protein-coding genes (based on Ensembl gene biotype annotations) and low-expressed genes (those with average values less than 1 in rlog scale for both treatments in comparison).
Data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75).
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: IGVtools tiled data file (TDF)

source_name: MV4;11 cultured cells

growth_protocol: MOLM13 or MV4;11 cells were cultured in RPMI1640 media supplemented with 10% Fetal bovine serum (v/v) (Gibco), 2 mM glutamine, 100U/mL penicillin (Gibco) and 100 ug/mL streptomycin (Gibco) and treated drug or DMSO-vehicle control

extract_protocol:
For RNA isolation, cells were lysed in 350 µl of RLT buffer and RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturers instructions for cell ine experiments. RNA samples from in vivo experiments were obtained by first isolating human cells from mouse marrow using the EasySep mouse-human chimera kit (Stem Cell Technologies) followed by RNA extraction using the PicoPure RNA isolation kit (Thermo Fisher).
Poly-A tail selection and library preparation were performed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA).
Sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) with 37bp, paired-end reads.

treatment_protocol: Treatment regimens indicated for each sample.

value: 3 days


title: MV411_RNA_len_5uM_VTP_50nM_3d_REP2_191024_TAATCG_S14

value: 5uM lenalidomide and 50nM VTP50469 combo


title: MV411_RNA_len_5uM_3d_REP3_191024_CACGAT_S6

value: 5uM lenalidomide


title: MV411_RNA_len_5uM_3d_REP1_191024_ACTGAT_S4


title: MV411_RNA_len_5uM_VTP_50nM_3d_REP3_191024_TACAGC_S15


title: MV411_RNA_VTP_50nM_3d_REP3_191024_GTGATC_S12


title: MV411_RNA_lenalidomide_d6_REP2_181206_CAACTA_S17

value: 6 days


title: MV411_RNA_VTP_50nM_3d_REP2_191024_CTATAC_S11


title: MV411_RNA_lenalidomide_d6_REP3_181206_CACCGG_S18


title: MV411_RNA_DMSO_d6_REP2_181206_ATGAGC_S14

value: DMSO


title: MV411_RNA_lenalidomide_d6_REP1_181206_CAAAAG_S16


title: MV411_RNA_DMSO_d6_REP1_181206_ACTGAT_S13


title: MV411_RNA_len_5uM_VTP_50nM_3d_REP1_191024_GACGAC_S13


title: MV411_RNA_DMSO_3d_REP2_191024_GTGAAA_S2


title: D26_C

value: WT


title: MV411_RNA_DMSO_3d_REP1_191024_CCGTCC_S1


title: MV411_RNA_CC220_1uM_VTP_50nM_3d_REP2_191024_TCATTC_S17

value: 1uM CC220 and 50nM VTP50469 combo


title: D19_C

value: Unknown


title: MV411_RNA_CC220_1uM_3d_REP3_191024_CGGAAT_S9

value: 1uM CC220


title: MV411_RNA_CC220_1uM_3d_REP1_191024_CATTTT_S7


title: MV411_RNA_CC220_1uM_VTP_50nM_3d_REP1_191024_TATAAT_S16


title: MV411_RNA_CC220_1uM_3d_REP2_191024_GGCTAC_S8


title: MOLM13_RNA_VTP_50nM_3d_REP3_191021_CTTGTA_S12

source_name: MOLM-13 cultured cells


title: MOLM13_RNA_VTP_50nM_3d_REP2_191021_GGCTAC_S11


title: MV411_RNA_len_5uM_3d_REP2_191024_CATGGC_S5


title: MV411_RNA_DMSO_3d_REP3_191024_GAGTGG_S3


title: MV411_RNA_DMSO_d6_REP3_181206_ATTCCT_S15


title: MOLM13_RNA_VTP_50nM_3d_REP1_191021_TAGCTT_S10


title: MOLM13_RNA_len_5uM_VTP_50nM_3d_REP2_191021_GAGTGG_S14


title: MOLM13_RNA_len_5uM_3d_REP3_191021_GCCAAT_S6


title: MOLM13_RNA_len_5uM_3d_REP2_191021_ACAGTG_S5


title: MOLM13_RNA_DMSO_3d_REP2_191021_CGATGT_S2


title: MOLM13_RNA_DMSO_3d_REP3_191021_TTAGGC_S3


title: MOLM13_RNA_CC220_1uM_VTP_50nM_3d_REP3_191021_TCGGCA_S18


title: MOLM13_RNA_len_5uM_VTP_50nM_3d_REP3_191021_ACTGAT_S15


title: MOLM13_RNA_len_5uM_3d_REP1_191021_TGACCA_S4


title: MOLM13_RNA_CC220_1uM_VTP_50nM_3d_REP1_191021_ATTCCT_S16


title: MOLM13_RNA_CC220_1uM_VTP_50nM_3d_REP2_191021_TCGAAG_S17


title: MOLM13_RNA_len_5uM_VTP_50nM_3d_REP1_191021_GTCCGC_S13


title: MV411_RNA_CC220_1uM_VTP_50nM_3d_REP3_191024_TCCCGA_S18


title: MOLM13_RNA_DMSO_3d_REP1_191021_ATCACG_S1


title: CBAM-68552_RNA_VTP_0.1pctfood_21d_REP5_191008_AGTTCC_S14

source_name: CBAM-68552 PDX cells extracted from mice

value: CBAM-68552 patient-derived xenograft

value: 21 days

value: 0.1% VTP50469 in chow


title: MOLM13_RNA_CC220_1uM_3d_REP1_191021_CAGATC_S7


title: CBAM-68552_RNA_VTP_0.1pctfood_21d_REP4_191008_AGTCAA_S13


title: MOLM13_RNA_CC220_1uM_3d_REP3_191021_GATCAG_S9


title: CBAM-68552_RNA_VTP_0.1pctfood_21d_REP2_191008_GGCTAC_S11


title: CBAM-68552_RNA_vehicle_21d_REP5_191008_ACAGTG_S5

value: vehicle control


title: CBAM-68552_RNA_VTP_0.1pctfood_21d_REP3_191008_CTTGTA_S12


title: CBAM-68552_RNA_vehicle_21d_REP4_191008_TGACCA_S4


title: CBAM-68552_RNA_vehicle_21d_REP3_191008_TTAGGC_S3


title: CBAM-68552_RNA_vehicle_21d_REP1_191008_ATCACG_S1


title: CBAM-68552_RNA_len_50mpk_21d_REP3_191008_ACTTGA_S8

value: 50mpk lenalidomide


title: CBAM-68552_RNA_len_50mpk_21d_REP4_191008_GATCAG_S9


title: MOLM13_RNA_CC220_1uM_3d_REP2_191021_ACTTGA_S8


title: CBAM-68552_RNA_len_50mpk_21d_REP1_191008_GCCAAT_S6


title: CBAM-68552_RNA_combo_21d_REP5_191008_ACTGAT_S19

value: 0.1% VTP50469 in chow and 50mpk lenalidomide


title: CBAM-68552_RNA_combo_21d_REP2_191008_GTCCGC_S16


title: CBAM-68552_RNA_combo_21d_REP4_191008_GAGTGG_S18


title: CBAM-68552_RNA_combo_21d_REP3_191008_GTGAAA_S17


title: MV411_ChIP_MLL1_steadystate_REP1_200713-200818

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human genome (GRCh37/hg19 Gencode r19) using STAR (v2.7.5a), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4). Final “deduped” .BAM files were indexed using SAMtools (v1.95).
ChIP-seq peaks were called using MACS2 (v2.1.4) with matched input samples as signal controls.
ChIP-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75) and ngs.plot (pileup heatmaps).
ChIP-seq MACS2 peak overlaps were determined using bedtools (intersect function; v2.28.0) and signal within peak regions determined using bedtools (coverage).
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: IGVtools tiled data file (TDF)

growth_protocol: MOLM13 or MV4;11 cells were cultured in RPMI1640 media supplemented with 10% Fetal bovine serum (v/v) (Gibco), 2 mM glutamine, 100U/mL penicillin (Gibco) and 100 ug/mL streptomycin (Gibco).

extract_protocol:
The cells were collected and washed twice in ice-cold PBS followed by crosslinking in 1% formaldehide (methanol free) for 5 minutes. Excess of formaldehyde was quenched by addition of Tris pH 8.0 to 100nM final and Glycine 125 mM final. Chromatin was cheared using Covaris E220 for 20 min with power 140; duty 5; bursts 200/sec. We then used 5ul aliquote of sherared chromatin to decrosslink in order to test completeness of shearing. Chromatin was considered passed QC if fragments 100-600bp were >90%. For immunoprecipitation we used chromatin from 20x10^6 cells, 5ug of specific antibody and Protein-A magnetic beads (Dynal). Following roatation at 4 degrees overnight, the immune complexes were wased in Mixed Micelle Buffer, buffer 500, LiCl/detergent solution and TE buffers. DNA was eluted from the beads using 100mM NaHCO3 100mM NaCl 1% SDS and purified using AmpureXP beads.
1-10 ng of ChIP'ed DNA was used for Illumina compatibl library generation using ThruPlex DNA-seq kit with 10 to 14 cycles of amplification with single or paired end barcodes

treatment_protocol: not applicable

value: anti-KMT2A/MLL1, rabbit

value: Bethyl


title: MV411_ChIP_MEIS1_steadystate_REP1_200713-200818

value: anti-MEIS1 rabbit, ChIP-grade

value: Abcam


title: MV411_ChIP_input_200717-200818

value: none (input)


title: CBAM-68552_RNA_VTP_0.1pctfood_21d_REP1_191008_TAGCTT_S10


title: CBAM-68552_RNA_len_50mpk_21d_REP2_191008_CAGATC_S7


title: MOLM13_ChIP_input_200717-200818


title: MV411_ChIP_IKZF1_steadystate_REP1_200713-200818

value: anti-IKAROS, rabbit monoclonal

value: Cell Signaling


title: MOLM13_ChIP_MLL1_steadystate_REP1_200713-200818


title: MV411_ChIP_MEN1_steadystate_REP1_200713-200818

value: anti-MENIN, rabbit


title: MOLM13_ChIP_IKZF1_steadystate_REP1_200713-200818


title: MOLM13_ChIP_MEIS1_steadystate_REP1_200713-200818


title: CBAM-68552_RNA_vehicle_21d_REP2_191008_CGATGT_S2


title: MV411_CUTRUN_H3K27ac_steadystate_REP1_200124_TCGGCA_S28

data_processing:
Library strategy: CUT&RUN
FASTQ files were processed using a version of the published CUT&RUNTools suite (Zhu et al., 2019), modified to run on a local SGE computer cluster.
Reads were trimmed using trimmomatic (v0.39) and aligned to the human genome (hg19) with Bowtie2 (v2.3.4.3).
Resulting BAM files were sorted, duplicates marked and filtered for fragments <120bp (all with SAMtools, v1.95), and peaks called (with MACS2, v2.1.4).
CUTRUN-seq MACS2 peak overlaps determined using bedtools (intersect; v2.28.0) and signal within peak regions determined using bedtools (coverage).
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: IGVtools tiled data file (TDF)

extract_protocol:
5x105 cells were washed twice in PBS, then once in a wash buffer (20mM HEPES pH 8.0, 150mM NaCl, 0.5mM spermidine) followed by immobilization on Concanavalin A magnetic beads activated in binding buffer (20mM HEPES pH 8.0, 10mM KCl, 1mM CaCl2, 1mM MnCl2) (Polyscience). The immobilized cells were permeabilized by washing in wash buffer supplemented with 0.1% Triton-X100 and incubated with anti-IKAROS antibody (Cell Signaling), rabbit IgG control (Bethyl), anti-CTCF (Cell Signaling) or anti-H3K27Ac (Diagenode) at 4C with addition of EDTA to 2mM. After 2-hour incubation the cells were washed twice and incubated with Protein-A-MNase conjugate at 4C. After 4 hours, the cells were washed twice and MNase was activated by addition of CaCl2 to 20mM. The DNA digestion was stopped by addition of EDTA and EGTA, both to 10mM. DNA fragments were collected in supernatant and purified using 1.8x AMPure XP beads (Beckman Coulter) which were then converted to Illumina compatible libraries using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England BioLabs)
NEBNext Ultra II DNA library prep kit for Illumina

treatment_protocol: Cell lines were treated in vitro with DMSO, CC220 1 uM, VTP-50469 50 nM or the drug combination for 72 hours.

value: 3 days

value: anti-Histone H3K27-acetyl

value: Diagenode


title: MV411_CUTRUN_IKZF1_DMSO_3d_REP1_200124_GACGAC_S21

value: DMSO


title: MV411_CUTRUN_IKZF1_CC220_1uM_3d_REP1_200124_TAATCG_S22

value: 1uM CC220


title: MV411_CUTRUN_IgG_steadystate_REP1_200124_TCGAAG_S27

value: Rabbit Isotype IgG control


title: MOLM13_ChIP_MEN1_steadystate_REP1_200713-200818


title: MOLM13_CUTRUN_IKZF1_VTP50469_50nM_3d_REP1_200124_GGCTAC_S11


title: MV411_CUTRUN_CTCF_steadystate_REP1_190522_CGTACG_S9

value: anti-CTCF (XP) R


title: MOLM13_CUTRUN_IKZF1_CC220_1uM_VTP_50nM_3d_REP1_200124_CTTGTA_S12

value: 1uM CC220 and 50nM VTP50469 combo


title: MOLM13_CUTRUN_IKZF1_DMSO_3d_REP1_200124_GATCAG_S9


title: CBAM-68552_RNA_combo_21d_REP1_191008_CCGTCC_S15

value: CBAM-68552 patient-derived xenograft

value: 21 days

value: 0.1% VTP50469 in chow and 50mpk lenalidomide


title: D109_M

value: Uterine fibroid patient

value: Myometrium

value: mut

value: HMB

value: Secretory


title: MOLM13_CUTRUN_H3K27ac_steadystate_REP1_200124_TCCCGA_S26


title: MOLM13_CUTRUN_CTCF_steadystate_REP1_190522_ATTCCT_S13

value: Cell Signaling


title: HCB1_SLAM_dVHL_60m_REP3_210720_TATGTC_S12

status: Public on Mar 03 2022

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
SLAMseq FASTQ files were analyzed using the Slamdunk pipeline, described elsewhere.
Differential SLAMseq expression was calculated using the BioConductor DESeq2 package (v1.24,0), using Slamdunk converted counts.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: SLAMdunk pipeline output (total and converted read counts per gene, Excel .xlsx) and DESeq2 rlog expression (CSV) and differential gene expression summary (Excel .xlsx)

molecule: polyA RNA

growth_protocol: The human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were maintained in IMDM with 20% FBS, 1xpenicillin/streptomycin (Gibco), 1xß-mercaptoethanol (Gibco), 6 µg/mL hIL3, 10 µg/mL hIL6, 10 µg/mL hSCF, 10 µg/mL TPO, and 10 µg/mL FLT3 (Stemcell Technologies) at 37°C and 5% CO2.

extract_protocol:
Cells were harvested at 300 g for 5 min at 4°C, washed with PBS and the RNA was extracted from the cell pellet using QIAshredders (Qiagen) and RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. 5ug of the extracted RNA was Iodoacetamide treated according to the SLAM-Seq Metabolic Labeling kit (Lexogen) manual instructions
Libraries were prepared using the QuantSeq 3’ mRNA-Seq Library Preparation kit (Lexogen)

treatment_protocol: Human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were plated at 5x106 cells/mL in the appropriate culture medium and were pre-treated with DMSO or dTAG-VHL (500nM) for 15min, 30min, 60min, or 120min. After the appropriate pre-treatment, cells were incubated with 4SU (Lexogen, SLAM-Seq Metabolic Labeling kit Lexogen) at a final concentration of 100μM and were incubated with continuous dTAG-VHL or DMSO control treatment for 60 min at 37°C / 5% CO2. Upon addition of 4SU, cells were kept away from light.

value: 60 minutes

value: 500nM dVHL


title: MV411_CUTRUN_IKZF1_VTP50469_50nM_3d_REP1_200124_TACAGC_S23

value: 3 days

value: anti-IKAROS, rabbit monoclonal


title: MOLM13_CUTRUN_IKZF1_CC220_1uM_3d_REP1_200124_TAGCTT_S10

value: 1uM CC220


title: HCB1_SLAM_dVHL_30m_REP2_210720_TTAACT_S8

value: 30 minutes


title: HCB1_SLAM_dVHL_30m_REP3_210720_ATACTG_S9


title: HCB1_SLAM_dVHL_15m_REP3_210720_GTCAGG_S6

value: 15 minutes


title: MV411_ATAC_steadystate_REP1_191120_TAAGGCGA_S1

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human genome (GRCh37/hg19 Gencode r19) using STAR (v2.7.5a), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4). Final “deduped” .BAM files were indexed using SAMtools (v1.95).
ATAC-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75).
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: IGVtools tiled data file (TDF), ABCenhancer enhancer loci (BED) and promoter associations (BEDPE)

extract_protocol:
Five million cells from each cell line (MV4;11 and MOLM13) were washed in PBS and resuspend in a hypotonic buffer (50 mM KCl, 10 mM MgSO4, 5 mM HEPES, 0.05% NP-40, 1 mM PMSF, 3 mM DTT) to generate intact nuclei and washed once in a nuclear wash buffer (10 mM Tris HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2). The resulting nuclei were counted and 50,000 nuclei per cell line were used for the transposition reaction. Nuclei were incubated in 1X TD Buffer (Illumina) and 2.5 µM of Transposase enzyme (Illumina) for 30 minutes at 37 °C. Then the transposed DNA was extracted from nuclei using the MiniElute PCR purification Kit (Qiagen). PCR reactions were carried out to amplify transposed DNA fragments and prep samples for sequencing using NEBNext High-Fidelity PCR kit (NEB) with a universal Nextera primer and a unique indexing primer for each cell line.
NEBNext® High-Fidelity PCR kit (NEB) with a universal Nextera primer

value: DMSO


title: MOLM13_ATAC_steadystate_REP1_191120_GGACTCCT_S5


title: HCB1_SLAM_dVHL_120m_REP3_210720_GAGTCC_S15

value: 120 minutes


title: MOLM13_CUTRUN_IgG_steadystate_REP1_200124_TCATTC_S25

value: Rabbit Isotype IgG control

value: Bethyl


title: HCB1_SLAM_dVHL_60m_REP1_210720_TGCACG_S10


title: MV411_CUTRUN_IKZF1_CC220_1uM_VTP_50nM_3d_REP1_200124_TATAAT_S24

value: 1uM CC220 and 50nM VTP50469 combo


title: HCB1_SLAM_dVHL_60m_REP2_210720_ATGAAC_S11


title: HCB1_SLAM_dVHL_120m_REP2_210720_CCTAAG_S14


title: HCB1_SLAM_dVHL_120m_REP1_210720_ACATTA_S13


title: HCB1_SLAM_dVHL_15m_REP2_210720_CGGTTA_S5


title: HCB1_SLAM_dVHL_30m_REP1_210720_ACCAGT_S7


title: HCB1_SLAM_DMSO_0m_REP2_210720_TCAGGA_S2

value: 0 minutes


title: HCB1_SLAM_DMSO_0m_REP1_210720_CAGCGT_S1


title: HCB1_SLAM_dVHL_15m_REP1_210720_GATCAC_S4


title: HCB1_SLAM_DMSO_0m_REP3_210720_TCTTAA_S3


title: HCB1_ChIP_MLL1c_dVHL_REP1_210928_TAGCTT_S3

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human(Gencode GRCh38/hg38) genome using STAR (v2.7.5a; params --alignIntronMax 1 --alignEndsType EndToEnd --alignM  atesGapMax 2000 for ChIPseq analysis), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
ChIP-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75) and ngs.plot (pileup heatmaps). ChIPseq peaks were called using MACS2 with appropriate input samples used as controls.
Gene body and TSS-associated signals were assessed using sitepro from the CEAS suite, using annotated gene body genomic intervals and TSS region intervals (-1kb to +3kb for each protein-coding transcript TSS, using gene orientation). A single representative TSS region was chosen for each gene, based on the site with the highest average signal in DMSO control samples.
For 3h treated sample interval counts were normalized using a ratio of TSS signal values among a set of selected housekeeping genes
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: IGVtools tiled data file (TDF); MACS2 peak output (tab-delimited, .xls); signal summary in TSS regions (Excel .xlsx)

extract_protocol:
The cells were collected and washed twice in ice-cold PBS followed by crosslinking in 1% formaldehide (methanol free) for 7-8 minutes. Excess of formaldehyde was quenched by addition of Tris pH 8.0 to 100nM final and Glycine 125 mM final. Chromatin was sheared using Covaris E220 for 30 min with power 140; duty 5; bursts 200/sec. We then used a 5ul aliquot of sherared chromatin to decrosslink in order to test completeness of shearing. Chromatin passed QC if fragments between 100-700bp were >90%. For immunoprecipitation we used chromatin from 20x10^6 cells, 5ug of specific antibody and Protein-A/G magnetic beads (Dynal). Following roatation at 4 degrees overnight, the immune complexes were wased in Mixed Micelle Buffer, buffer 500, LiCl/detergent solution and TE buffers. DNA was eluted from the beads using 100mM NaHCO3 100mM NaCl 1% SDS and purified using AmpureXP beads.
1-10 ng of ChIP'ed DNA was used for the library preparation using the SMARTer ThruPLEX DNA-Seq Kit (Takara)

treatment_protocol: Treatment regimen and timepoint indicated for each sample. In brief, human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were treated with 500nm dTAG-VHL or DMSO control for 3 hours and  ChIPs were performed as indicated.

value: MLL1 (c terminus)

value: Active Motif

value: 3 hours


title: hsCB-FKBP12-MA9_ChIP_MLL1_dVHL_500nM_3h_REP1_190814_TACAGGAT_S27

value: MLL1 (N terminus)


title: hsCB-FKBP12-MA9_ChIP_MLL1_DMSO_3h_REP1_190814_TAGTGACT_S26


title: HCB1_ChIP_MLL1c_DMSO_REP1_210928_GATCAG_S2


title: HCB1_RNA_combo_2500nM-100nM_1d_REP3_1907_combined_TATAAT_S40

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human (Gencode GRCh38/hg38) genome using STAR (v2.7.5a, sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
RNA-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75).
Raw per-gene counts calculated with HTSeq (htseq-count, v0.6.1pl).
Differential RNA-seq expression was calculated using the BioConductor DESeq2 package (v1.24,0), using raw unnormalized per-gene counts from deduplicated BAMs.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: IGVtools tiled data file (TDF), HTSeq per-gene read counts (TXT), DESeq2 differentially expressed gene summary (CSV)

growth_protocol: Human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were maintained in IMDM with 20% FBS, 1xpenicillin/streptomycin (Gibco), 1xß-mercaptoethanol (Gibco), 6 µg/mL hIL3, 10 µg/mL hIL6, 10 µg/mL hSCF, 10 µg/mL TPO, and 10 µg/mL FLT3 (Stemcell Technologies) at 37°C and 5% CO2

treatment_protocol: Treatment regimen and timepoint indicated for each sample. In brief, human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were treated with 250nM EPZ-5676 and 100nM VTP-50469 for 24 hours.

value: 1 day


title: HCB1_ChIP_input_steadystate_REP1_210928_ACTTGA_S1


title: HCB1_RNA_combo_2500nM-100nM_1d_REP2_1907_combined_CACCGG_S25


title: Armstrong001_PR575_30m_2

description: nascent RNA

data_processing:
Library strategy: PRO-seq
FASTQ read pairs were trimmed to 41bp per mate, and read pairs with a minimum average base quality score of 20 retained. Read pairs were further trimmed using cutadapt 1.14 to remove adapter sequences and low-quality 3’ bases (--match-read-wildcards -m 20 -q 10).
R1 reads, corresponding to RNA 3’ ends, were then aligned to the spiked in Drosophila genome index (dm3) using Bowtie 1.2.2 (-v 2 -p 6 --best --un), with those reads not mapping to the spike genome serving as input to the primary genome alignment step (using Bowtie 1.2.2 options -v 2 --best). Reads mapping to the hg38 reference genome were then sorted, via samtools 1.3.1 (-n), and subsequently converted to bedGraph format using a custom script (bowtie2stdBedGraph.pl).
For promoter reads, annotated transcription start sites were obtained from human (GRCh38.99) GTFs from Ensembl. After removing transcripts with {immunoglobulin, Mt, Mt_tRNA, rRNA} biotypes, PRO-seq signal in each sample was calculated in the window from the annotated TSS to +150 nt downstream, using a custom script, make_heatmap.pl.
For differential expression analysis, reads were summed within the TSS to TES window for each active gene.  DEseq2, using the Wald test, was used to determine statistically significant differentially expressed genes. Unless otherwise noted, the default size factors determined by DEseq2 were used.
Pausing indices were generated by summing in the window from TSS to +150 for each active gene, and from +250 downstream of the TSS to the TES, using the refined annotations described above. The ratio of promoter proximal signal (TSS to +150) over gene body signal (+250 to TES) is the Pausing Index.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: DESeq2 analysis summary (Excel .xlsx)

extract_protocol:
10 million cells were harvested and washed once with ice cold PBS, cell pellets were resuspended in 250uL wash buffer to get single-cell suspensions. 10mL permealization buffer was added and samples were incubated on ice for 5 min. Cells were spun down and pellets were washed in 10mL wash buffer. Cells were spun again and resuspended in freezing buffer. Cells were counted and permealization was visualized using tryphan blue. 5 million permealized cells were snap-frozen in 500ul freezing buffer in liquid nitrogen and stored at -80°C. All subsequent steps were performed by the Nascent Transcriptomics Core at Harvard Medical School, Boston, MA. In brief, run-on reactions were performed at 37°C.  Adenylated 3' adapter was prepared using the 5' DNA adenylation kit (NEB) and ligated using T4 RNA ligase 2, truncated KQ (NEB, per manufacturers instructions with 15% PEG-8000 final) and incubated at 16°C overnight.  180uL of betaine blocking buffer with 0.6 uM blocking oligo was mixed with ligations and incubated for 5 min at 65°C and 2 min on ice prior to addition of streptavidin beads.  After T4 polynucleotide kinase (NEB) treatment, beads were washed once each with high salt, low salt, and blocking oligo wash solutions and resuspended in 5' adapter mix.
Eluted cDNA was amplified 5-cycles (NEBNext Ultra II Q5 master mix (NEB)) with Illumina TruSeq PCR primers RP-1 and RPI-X following the manufacturer's suggested cycling protocol for library construction. A portion of preCR was serially diluted and for test amplification to determine optimal amplification of final libraries.

treatment_protocol: Human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were plated at 5x106 cells/mL in the appropriate culture medium and were treated with DMSO or dTAG-VHL (500nM) for 15min, 30min, or 180min. After the appropriate treatment, 10 million cells were harvested and permealized as indicated

value: 500nM dVHL


title: Armstrong001_PR576_3h_2

description: nascent RNA


title: HCB1_RNA_combo_2500nM-100nM_1d_REP1_1907_combined_TAGCTT_S10


title: Armstrong001_PR570_15m_1

description: nascent RNA


title: Armstrong001_PR569_untreated_1

description: nascent RNA

value: untreated


title: HCB1_ATAC_untreated_0m_REP1_2107x2_AGGCAGAA_S1

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human(Gencode GRCh38/hg38) genome using STAR (v2.7.5a; params --alignIntronMax 1 --alignEndsType EndToEnd --alignM  atesGapMax 2000 for ChIPseq analysis), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
ATAC-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75). ATACseq peaks were called using MACS2 independently for each sample.
Gene body and TSS-associated signals were assessed using sitepro from the CEAS suite, using annotated gene body genomic intervals and TSS region intervals (-1kb to +3kb for each protein-coding transcript TSS, using gene orientation). A single representative TSS region was chosen for each gene, based on the site with the highest average signal in DMSO control samples.
For treated samples interval counts were normalized using a ratio of TSS signal values among a set of selected housekeeping genes
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: IGVtools tiled data file (TDF), MACS2 peak calls (tab-delimited; .xls), CEAS sitepro TSS signal summary (Excel .xlsx)

extract_protocol:
Cells were harvested and washed in PBS, pellets were then resuspended in lysis buffer (10 mM Tris-HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl2; 0.1% IGEPAL CA-630). Cells were spun down immediately at 500 × g for 10 min at 4 °C. Following this, the pellet was then resuspended in a transposase reaction mixture (2X TD buffer, 20X transposase) (Illumina) at 37 °C for 30 minutes. DNA was purified using a MinElute kit (Qiagen) as per the manufacturer’s instructions.
The DNA fragments were amplified in a PCR reaction with 2X NEBNext High-Fidelity PCR Master Mix (New England Biolabs) for 11 cycles and purified using AMPure XP beads (Beckman Coulter)

treatment_protocol: Human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were plated at 5x106 cells/mL in the appropriate culture medium and were treated with DMSO or dTAG-VHL (500nM) for 180min.


title: Armstrong001_PR572_3h_1

description: nascent RNA


title: Armstrong001_PR574_15m_2

description: nascent RNA


title: Armstrong001_PR573_untreated_2

description: nascent RNA


title: Armstrong001_PR571_30m_1

description: nascent RNA


title: HCB1_ATAC_untreated_0m_REP2_2107x2_TCCTGAGC_S2


title: HCB1_ATAC_dVHL_180m_REP2_2107x2_AGGTTGGG_S12

value: 180 minutes


title: HCB1_ChIP_input_DMSO_24h_REP1_190814_TTCCTGCT_S25

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human(Gencode GRCh38/hg38) genome using STAR (v2.7.5a; params --alignIntronMax 1 --alignEndsType EndToEnd --alignM  atesGapMax 2000 for ChIPseq analysis), sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
ChIP-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75) and ngs.plot (pileup heatmaps). ChIPseq peaks were called using MACS2 with appropriate input samples used as controls.
Gene body and TSS-associated signals were assessed using sitepro from the CEAS suite, using annotated gene body genomic intervals and TSS region intervals (-1kb to +3kb for each protein-coding transcript TSS, using gene orientation). A single representative TSS region was chosen for each gene, based on the site iwth the highest average signal in DMSO control samples.
For 3h and 24h samples, treated sample interval counts were normalized using a ratio of TSS signal values among a set of selected housekeeping genes; for later timepoints, reads were normalized using ratios of total read counts between treated and control sample pairs.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: IGVtools tiled data file (TDF); MACS2 peak output (tab-delimited, .xls); signal summary in TSS and gene body regions (Excel .xlsx)

growth_protocol: The human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were maintained in IMDM with 20% FBS, 1xpenicillin/streptomycin (Gibco), 1xß-mercaptoethanol (Gibco), 6 µg/mL hIL3, 10 µg/mL hIL6, 10 µg/mL hSCF, 10 µg/mL TPO, and 10 µg/mL FLT3 (Stemcell Technologies) at 37°C and 5% CO2.  MOLM13 cells were cultured in RPMI1640 media supplemented with 10% Fetal bovine serum (v/v) (Gibco), 2 mM glutamine, 100U/mL penicillin (Gibco) and 100 ug/mL streptomycin (Gibco).

treatment_protocol: Treatment regimen and timepoint indicated for each sample. In brief, human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were treated with 500nm dTAG-VHL or DMSO control for 3 hours or 24 hours and  ChIPs were performed as indicated. The human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were also treated with DMSO control, 250nM EPZ-5676, 100nM VTP-50469, or the EPZ/VTP combination for 4 days, and  ChIPs  were performed as  indicated. MOLM13 cells were treated with either DMSO control, 1uM EPZ-5676 , 100nM VTP-50469 or the combination for 4 days and ChIPs were performed against MLL1 and H3K27ac.

value: 24 hours


title: MOLM13_ChIP_MLL1_VTP_100nM_d4_REP1_201007_GTCCGC_S8

value: 4 days


title: MOLM13_ChIP_input_DMSO_d4_REP1_201007_CGATGT_S1


title: MOLM13_ChIP_MLL1_EPZ_1uM_VTP_100nM_d4_REP1_201007_GTGAAA_S9


title: MOLM13_ChIP_MLL1_DMSO_d4_REP1_201007_ATGTCA_S6


title: MOLM13_ChIP_H3K27ac_EPZ_1uM_VTP_100nM_d4_REP1_201007_GCCAAT_S5

value: Diagenode


title: MOLM13_ChIP_H3K27ac_VTP_100nM_d4_REP1_201007_ACAGTG_S4


title: MOLM13_ChIP_MLL1_EPZ_1uM_d4_REP1_201007_CCGTCC_S7

value: 1uM EPZ5676


title: HCB1_ATAC_dVHL_180m_REP1_2107x2_GAGGGGTT_S11


title: HCB1_ChIP_input_DMSO_24h_REP1_190814_TGCGATCT_S24

value: DMSO


title: MOLM13_ChIP_H3K27ac_EPZ_1uM_d4_REP1_201007_TGACCA_S3


title: HCB1_ChIP_HA_dVHL_500nM_24h_REP1_190814_ACTTGATG_S7

value: HA

value: CST


title: HCB1_ChIP_HA_dVHL_500nM_3h_REP1_190814_GCCAATGT_S5


title: HCB1_ChIP_H3K9ac_dVHL_500nM_3h_REP1_190814_TAGCTTGT_S9

value: Abcam


title: HCB1_ChIP_HA_DMSO_24h_REP1_190814_CAGATCTG_S6


title: HCB1_ChIP_HA_DMSO_3h_REP1_190814_ACAGTGGT_S4


title: HCB1_ChIP_H3K9ac_dVHL_500nM_24h_REP1_190814_CTTGTACT_S11


title: HCB1_ChIP_H3K9ac_DMSO_24h_REP1_190814_GGCTACAG_S10


title: HCB1_ChIP_H3K9ac_DMSO_3h_REP1_190814_GATCAGCG_S8


title: HCB1_ChIP_H3K79me2_dVHL_500nM_3h_REP1_190814_TGATACGT_S21


title: MOLM13_ChIP_H3K27ac_DMSO_d4_REP1_201007_TTAGGC_S2


title: HCB1_ChIP_H3K79me2_dVHL_500nM_24h_REP1_190814_GGTTGGAC_S23


title: HCB1_ChIP_H3K27ac_DMSO_24h_REP1_190814_TCTGCTGT_S18


title: HCB1_ChIP_H3K27ac_dVHL_500nM_3h_REP1_190814_TTCTGTGT_S17


title: HCB1_ChIP_H3K27ac_dVHL_500nM_24h_REP1_190814_TTGGAGGT_S19


title: HCB1_ChIP_RNAPol2_pS5_dVHL_500nM_3h_REP1_1909x2_TTCCATTG_S32


title: HCB1_ChIP_RNAPol2_pS5_DMSO_3h_REP1_1909x2_TAGTCTTG_S31


title: HCB1_ChIP_RNAPol2_pS2_dVHL_500nM_3h_REP1_1909x2_TACAGGAT_S28


title: HCB1_ChIP_RNAPol2_pS2_DMSO_3h_REP1_1909x2_TAGTGACT_S27


title: HCB1_ChIP_input_DMSO_4d_REP1_1912x2_TTAGGCAT_S3


title: HCB1_ChIP_RNAPol2_dVHL_500nM_3h_REP1_1909x2_GGTTGGAC_S24


title: HCB1_ChIP_input_DMSO_4d_REP1_200908_CGATGT_S2

description:
TSS_signal_Figure5.xlsx
TSS_signal_HA_ChIPs.xlsx


title: HCB1_ChIP_input_DMSO_d4_REP1_201007_ATCACG_S1


title: HCB1_ChIP_MEN1_DMSO_3h_REP1_1912x2_TCCGTCTT_S16

value: Bethyl


title: HCB1_ChIP_MEN1_dVHL_500nM_3h_REP1_1912x2_TGTACCTT_S17


title: HCB1_ChIP_RNAPol2_DMSO_3h_REP1_1909x2_GTGCTACC_S23


title: HCB1_ChIP_input_DMSO_3h_REP2_1909x2_TAGCTTGT_S10


title: HCB1_ChIP_input_DMSO_3h_REP1_200211_ACTTGA_S8


title: HCB1_ChIP_input_DMSO_3h_REP1_200721_TAGTGACT_S3


title: HCB1_ChIP_Input_DMSO_3h_REP1_191121_ATCACGTT_S1


title: HCB1_ChIP_HA_VTP_100nM_4d_REP1_200908_AGTTCC_S14


title: HCB1_ChIP_input_DMSO_24h_REP1_200721_TACAGGAT_S4


title: HCB1_ChIP_input_DMSO_3h_REP1_1912x2_ATCACGTT_S1


title: HCB1_ChIP_H3K79me2_DMSO_24h_REP1_190814_GTGCTACC_S22


title: HCB1_ChIP_H3K79me2_DMSO_3h_REP1_190814_TCGAGCGT_S20


title: HCB1_ChIP_H3K27ac_DMSO_3h_REP1_190814_TGTACCTT_S16


title: HCB1_ChIP_HA_VTP_100nM_4d_REP1_1912x2_TAAGCGTT_S15


title: HCB1_ChIP_HA_EPZ_250nM_4d_REP1_1912x2_TCTCGGTT_S14


title: HCB1_ChIP_HA_EPZ_250nM_4d_REP1_200908_AGTCAA_S13


title: HCB1_ChIP_HA_EPZ_250nM_VTP_100nM_4d_REP1_200908_ATGTCA_S15


title: HCB1_ChIP_HA_dVHL_500nM_4d_REP1_200908_CTTGTA_S12


title: HCB1_ChIP_HA_dVHL_500nM_4d_REP1_1912x2_TGGTTGTT_S13

description:
TSS_signal_Figure4.xlsx
TSS_signal_HA_ChIPs.xlsx


title: HCB1_ChIP_HA_DMSO_4d_REP1_200908_GTGGCC_S19


title: HCB1_ChIP_HA_dVHL_500nM_3h_REP1_1909x2_TAAGTTCG_S40


title: HCB1_ChIP_HA_DMSO_4d_REP1_1912x2_CTTGTACT_S12


title: HCB1_ChIP_H3K27ac_VTP_100nM_d4_REP1_201007_CTTGTA_S5


title: HCB1_ChIP_H3K4me3_DMSO_3h_REP1_200721_TCCTCAAT_S5


title: HCB1_ChIP_H3K27ac_EPZ_1uM_VTP_100nM_d4_REP1_201007_AGTTCC_S6


title: HCB1_ChIP_H3K27ac_dVHL_500nM_d4_REP1_201007_GATCAG_S3


title: HCB1_ChIP_CDK9_dVHL_500nM_3h_REP1_200211_CTTGTA_S12


title: HCB1_ChIP_H3K27ac_DMSO_d4_REP1_201007_CAGATC_S2


title: HCB1_ChIP_H3K4me3_DMSO_24h_REP1_200721_TAGTCTTG_S7


title: HCB1_ChIP_CDK9_DMSO_3h_REP1_200211_GGCTAC_S11


title: HCB1_ChIP_AFF4_dVHL_500nM_3h_REP1_191121_TTAGGCAT_S3


title: HCB1_ChIP_DOT1L_DMSO_3h_REP1_1909x2_TCGAGCGT_S21


title: HCB1_ChIP_DOT1L_dVHL_500nM_3h_REP1_1909x2_TGATACGT_S22


title: HCB1_ChIP_AFF4_DMSO_3h_REP1_191121_CGATGTTT_S2


title: MOLM13_RNA_EPZ_1uM_VTP_100nM_5d_REP3_210312_TCGGCA_S12

description: MOLM13_EPZ_VTP_combo_vs_untreated_5d_DEseq2_Diffexp_allGenes_allSamples_rmdup-20210315.csv

data_processing:
Raw Illumina sequencer output was converted to FASTQ format using bcl2fastq (v2.20.0.422).
Reads (paired-end 37-mers) were aligned to the human (Gencode GRCh38/hg38) or mouse (Gencode M24/mm10) genome using STAR (v2.7.5a, sorted and duplicates marked/removed with picard pipeline tools (v2.9.4).
Final “deduped” .BAM files were indexed using SAMtools (v1.95).
RNA-seq data visualizations were produced using IGVtools (TDF signal pileups; v2.3.75).
Raw per-gene counts calculated with HTSeq (htseq-count, v0.6.1pl).
Differential RNA-seq expression was calculated using the BioConductor DESeq2 package (v1.24,0), using raw unnormalized per-gene counts from deduplicated BAMs.
Genome_build: GRCh38/hg38 and M24/mm10
Supplementary_files_format_and_content: IGVtools tiled data file (TDF), HTSeq per-gene read counts (TXT), DESeq2 differentially expressed gene summary (CSV)

growth_protocol: Murine MLL-AF9-HA-FKBP12 transformed leukemic cells (BM2222) were maintained in IMDM with 20% FBS, 1xpenicillin/streptomycin (Gibco), 1xß-mercaptoethanol (Gibco), 6 µg/mL mIL3, 10 µg/mL mIL6, and 20 µg/mL mSCF (Stemcell Technologies) at 37°C and 5% CO2. The human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were maintained in IMDM with 20% FBS, 1xpenicillin/streptomycin (Gibco), 1xß-mercaptoethanol (Gibco), 6 µg/mL hIL3, 10 µg/mL hIL6, 10 µg/mL hSCF, 10 µg/mL TPO, and 10 µg/mL FLT3 (Stemcell Technologies) at 37°C and 5% CO2.  MOLM13 cells were cultured in RPMI1640 media supplemented with 10% Fetal bovine serum (v/v) (Gibco), 2 mM glutamine, 100U/mL penicillin (Gibco) and 100 ug/mL streptomycin (Gibco). The murine neutrophils were not maintained ex vivo.

extract_protocol:
For RNA isolation from cell lines, 1x10^6 cells were lysed in 350 µl of RLT buffer and RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturers instructions for cell ine experiments. RNA samples from the murine neutrophils were obtained by isolating neutrophils from mouse marrow using the EasySep™ Mouse Neutrophil Enrichment Kit according to the manufacturer’s instructions (Stem Cell Technologies). The neutrophils were then lysed in 350 µl of RLT buffer and RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturers instructions for cell ine experiments.
Poly-A tail selection and library preparation were performed using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA).
Sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) with 37bp, paired-end reads.

treatment_protocol: Treatment regimen and timepoint indicated for each sample. In brief, murine MLL-AF9-HA-FKBP12 transformed leukemic cells (BM2222) were treated with 500nm dTAG-13 or DMSO control for 3 hours, 24 hours or 5 days. The human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were treated with 500nm dTAG-VHL or DMSO control for 3 hours, 24 hours or 5 days. The human CD34+ MLL-AF9-HA-FKBP12 transformed cells (HCB1) were also treated with DMSO control, 250nM EPZ-5676, or 100nM VTP-50469 for 24 hours, 3 days, and 5 days, as well as with the EPZ/VTP combination for 5 days. MOLM13 cells were treated with either DMSO control, 1uM EPZ-5676 , 100nM VTP-50469 or the combination for five days. The murine neutrophils were isolated from mice without any drug treatments.

value: 5 days


title: MOLM13_RNA_VTP_100nM_5d_REP1_210312_TACAGC_S7


title: MOLM13_RNA_EPZ_1uM_VTP_100nM_5d_REP1_210312_TCCCGA_S10


title: MOLM13_RNA_EPZ_1uM_5d_REP3_210312_TAATCG_S6


title: MOLM13_RNA_EPZ_1uM_5d_REP2_210312_GACGAC_S5


title: HCB1_ChIP_HA_DMSO_3h_REP1_1909x2_TCAGGAGG_S39


title: MOLM13_RNA_VTP_100nM_5d_REP3_210312_TCATTC_S9


title: MOLM13_RNA_EPZ_1uM_5d_REP1_210312_GTGATC_S4


title: MOLM13_RNA_DMSO_5d_REP2_210312_CTAGCT_S2


title: HCB1_ChIP_H3K4me3_dVHL_500nM_3h_REP1_200721_TGTGGTTG_S6


title: HCB1_ChIP_H3K4me3_dVHL_500nM_24h_REP1_200721_TTCCATTG_S8


title: MOLM13_RNA_DMSO_5d_REP3_210312_CTATAC_S3


title: HCB1_RNA_VTP_100nM_72h_REP3_2007x2_TATAAT_S44

description: HCB1_72h_EPZ_VTP_combo_dVHL_vs_untreated_DESeq2_DiffExp_allGenes_allSamples_rmdup-20200727.csv

value: 72 hours


title: MOLM13_RNA_EPZ_1uM_VTP_100nM_5d_REP2_210312_TCGAAG_S11


title: MOLM13_RNA_DMSO_5d_REP1_210312_CGGAAT_S1


title: HCB1_RNA_VTP_100nM_72h_REP2_2007x2_CTATAC_S39


title: HCB1_RNA_VTP_100nM_72h_REP1_2007x2_CATGGC_S34


title: HCB1_ChIP_H3K27ac_EPZ_1uM_d4_REP1_201007_GGCTAC_S4


title: HCB1_RNA_VTP_100nM_5d_REP2_1907_combined_CAGGCG_S29

description: HCB1_5d_EPZ_VTP_combo_dVHL_vs_untreated_newDMSO5d_DESeq2_DiffExp_allGenes_allSamples_rmdup-20200731.csv


title: HCB1_RNA_VTP_100nM_5d_REP1_1907_combined_AGTTCC_S14


title: HCB1_RNA_VTP_100nM_5d_REP3_1907_combined_TCGAAG_S43


title: MOLM13_RNA_VTP_100nM_5d_REP2_210312_TATAAT_S8


title: HCB1_RNA_EPZ_250nM_VTP_100nM_72h_REP2_2007x2_GTGATC_S40


title: HCB1_RNA_EPZ_250nM_VTP_100nM_72h_REP1_2007x2_CATTTT_S35


title: HCB1_RNA_VTP_100nM_1d_REP3_1907_combined_TACAGC_S39

description: HCB1_1d_EPZ_VTP_dVHL_vs_untreated_DESeq2_DiffExp_allGenes_allSamples_rmdup-20200731.csv


title: HCB1_RNA_VTP_100nM_1d_REP1_1907_combined_GATCAG_S9


title: HCB1_RNA_EPZ_250nM_72h_REP2_2007x2_CTAGCT_S38


title: HCB1_RNA_EPZ_2500nM_5d_REP3_1907_combined_TCCCGA_S42


title: HCB1_RNA_EPZ_2500nM_1d_REP2_1907_combined_ACTGAT_S23


title: HCB1_RNA_EPZ_2500nM_5d_REP2_1907_combined_CACTCA_S28


title: HCB1_RNA_EPZ_2500nM_1d_REP1_1907_combined_ACTTGA_S8


title: HCB1_RNA_EPZ_250nM_72h_REP1_2007x2_CAGGCG_S33


title: HCB1_RNA_dVHL_500nM_72h_REP1_2007x2_CACTCA_S32


title: HCB1_RNA_dVHL_500nM_72h_REP2_2007x2_CGGAAT_S37


title: HCB1_RNA_dVHL_500nM_5d_REP1_1907_combined_CTTGTA_S12


title: HCB1_RNA_dVHL_500nM_3h_REP3_1907_combined_CCAACA_S32

description: HCB1_3h_dVHL_vs_untreated_DESeq2_DiffExp_allGenes_allSamples_rmdup-20200731.csv

value: 3 hours


title: HCB1_RNA_dVHL_500nM_5d_REP3_1907_combined_TCATTC_S41


title: HCB1_RNA_dVHL_500nM_3h_REP1_1907_combined_CGATGT_S2


title: HCB1_RNA_EPZ_250nM_VTP_100nM_72h_REP3_2007x2_TCATTC_S45


title: HCB1_RNA_VTP_100nM_1d_REP2_1907_combined_CAACTA_S24


title: HCB1_RNA_dVHL_500nM_5d_REP2_1907_combined_CACGAT_S27


title: HCB1_RNA_dVHL_500nM_1d_REP1_1907_combined_CAGATC_S7


title: HCB1_RNA_dVHL_500nM_3h_REP2_1907_combined_GTCCGC_S17


title: HCB1_RNA_dVHL_500nM_1d_REP2_1907_combined_GAGTGG_S22


title: HCB1_RNA_DMSO_72h_REP2_2007x2_CCAACA_S36


title: HCB1_RNA_EPZ_250nM_72h_REP3_2007x2_TACAGC_S43


title: HCB1_RNA_DMSO_72h_REP3_2007x2_GACGAC_S41


title: HCB1_RNA_DMSO_72h_REP1_2007x2_CACGAT_S31


title: HCB1_RNA_DMSO_5d_REP2_1907_combined_ATTCCT_S26


title: HCB1_RNA_DMSO_5d_REP1_2007x2_TCCCGA_S46


title: HCB1_RNA_DMSO_5d_REP1_1907_combined_GGCTAC_S11


title: HCB1_RNA_DMSO_3h_REP3_1907_combined_CATTTT_S31


title: HCB1_RNA_EPZ_2500nM_1d_REP3_1907_combined_TAATCG_S38


title: HCB1_RNA_EPZ_2500nM_5d_REP1_1907_combined_AGTCAA_S13


title: HCB1_RNA_DMSO_3h_REP2_1907_combined_CCGTCC_S16


title: HCB1_RNA_dVHL_500nM_72h_REP3_2007x2_TAATCG_S42


title: HCB1_RNA_DMSO_3h_REP1_1907_combined_ATCACG_S1


title: HCB1_RNA_DMSO_1d_REP2_1907_combined_CGTACG_S21


title: HCB1_RNA_combo_2500nM-100nM_5d_REP3_1907_combined_TCGGCA_S44


title: HCB1_RNA_DMSO_1d_REP1_1907_combined_GCCAAT_S6


title: HCB1_RNA_DMSO_1d_REP3_1907_combined_GTGATC_S36


title: HCB1_RNA_combo_2500nM-100nM_5d_REP2_1907_combined_CATGGC_S30


title: mouse_RNA_neutrophils_steadystate_mouse3_180927_ATGTCA_S15

value: neutrophils


title: mouse_RNA_neutrophils_steadystate_mouse2_180927_AGTTCC_S14


title: HCB1_RNA_combo_2500nM-100nM_5d_REP1_1907_combined_ATGTCA_S15


title: HCB1_RNA_dVHL_500nM_1d_REP3_1907_combined_GACGAC_S37

value: 1 day


title: BM2222_RNA_dTAG_500nM_5d_REP3_CTAGCT_S38

description: BM2222_dTAG_vs_untreated_3h_1d_5d_DESeq2_DiffExp_allGenes_allSamples_rmdup_ERCCSizeEst-20181024.csv

value: 500nM dTAG13


title: BM2222_RNA_dTAG_500nM_5d_REP1_TAGCTT_S10


title: mouse_RNA_neutrophils_steadystate_mouse1_180927_AGTCAA_S13


title: BM2222_RNA_dTAG_500nM_3h_REP3_CACCGG_S30


title: BM2222_RNA_dTAG_500nM_3h_REP2_GTAGAG_S17


title: BM2222_RNA_dTAG_500nM_1d_REP2_GTTTCG_S21


title: BM2222_RNA_dTAG_500nM_1d_REP3_CATGGC_S34


title: BM2222_RNA_dTAG_500nM_5d_REP2_ACTGAT_S25


title: BM2222_RNA_dTAG_500nM_3h_REP1_CGATGT_S2


title: BM2222_RNA_DMSO_5d_REP3_CGGAAT_S37


title: BM2222_RNA_DMSO_5d_REP1_GATCAG_S9


title: BM2222_RNA_DMSO_5d_REP2_GGTAGC_S24


title: BM2222_RNA_dTAG_500nM_1d_REP1_GCCAAT_S6


title: BM2222_RNA_DMSO_3h_REP2_CCGTCC_S16


title: BM2222_RNA_DMSO_3h_REP1_ATCACG_S1


title: BM2222_RNA_DMSO_1d_REP3_CAGGCG_S33


title: BM2222_RNA_DMSO_3h_REP3_CAACTA_S29


title: C_genomic_input

status: Public on Jan 27 2022

city: New York

first: Aarthi

last: Ramakrishnan

state: New York

address: 1425 Madison Avenue

department: Neuroscience

institute: Icahn School of Medicine at Mount Sinai

data_processing:
Quality controls using FastQC (v0.11.2) were generated and raw sequencing reads were then aligned to the mouse mm10 genome using default settings of Bowtie (v2.2.0).
Peak-calling was performed using MACS (v2.1.1) (Zhang et al., 2008) with various P-value cutoffs. The peak bed files were generated from peak calling against genomic input control or IgG control with the default setting (10-5 cutoff).
The common peaks from these two bed files were used for subsequent analyses. The overlapping peaks of bed files were identified using the bedtools from Galaxy (https://usegalaxy.org/) (Afgan et al., 2018).
Genome_build: mm10
Supplementary_files_format_and_content: bed

source_name: Kidney

extract_protocol:
Kidneys from E13.5-14.0 Eya1Six1 embryos were collected for ChIP-seq assay. ChIP-seq was performed according to previous protocols with some modifications (Li et al., 2020, Xu et al., 2021). Briefly, the kidneys were cross-linked, homogenized and lysed and chromatin was sonicated as described (Li et al., 2021). Sonicated chromatin was cleared by pelleting insoluble material at 13,000 RPM at 4°C, followed by preclear with protein A/G beads and incubation with 1~2 mg anti-Six1 overnight or 1~2 mg rabbit IgG as a negative control.
The pulldown and input control sequencing libraries were generated using the ThruPLEX DNA-seq Kit (R400429, Rubicon Genomics) and sequenced on Illumina NextSeq 500.

tag: strain background

value: mixed; C57BL/6 and 129SV

tag: genotype

value: Kidney

value: Input


title: ND566.Day24_Continuous.Sox4_KO

status: Public on Dec 06 2021

city: Philadelphia

first: Gregory

last: Donahue

state: PA

address: 3400 Civic Center Blvd, Bldg 421

department: Cell & Developmental Biology

institute: The University of Pennsylvania

data_processing:
Library strategy: RNA-Seq
Alignment: 10X v2 sequencing data were aligned to the GRCh38 genome, filtered, and then barcodes and unique molecular identifiers were counted using the Cell Ranger v3.1.0 command cellranger count.
Genome_build: GRCh38
Supplementary_files_format_and_content: We include cloupe files (the output of cellranger count) which can be loaded in the Loupe Browser to see the expression level of genes in each cell.

source_name: M5CAR T cells (PBMCs)

growth_protocol: Primary human CD4+ T and CD8+ T cells from normal donors were provided by University of Pennsylvania Human Immunology Core. CAR T cells were generated as previously described (Carpenito et al., 2009). Briefly, CD4+ and CD8+ T at 1:1 ratio at 1 x 10e6 cells/ml were activated with Dynabeads CD3/CD28 CTSTM (Gibco, Life Technologies) at a 3:1 bead-to-cell ratio. Approximately after 24 hours, T cells were transduced at a multiplicity of infection (MOI) of 5. At day 5 beads were removed from cultures. T cell cultures were maintained at 8 x 10e5 cells/ml. Cell number and volume were monitored daily using Multisizer 4 Coulter Counter (Beckmanwaslter). Transduced T cells were cryopreserved when reached the resting state, as determined by cell size.

extract_protocol:
For flow cytometry and sorting assays of CAE, M5 CAR T cells were stained in fluorescence-activated cell sorting (FACS) buffer consisting of PBS (Gibco), 0.5% bovine serum albumin (BSA) (GEMINI), 2 mM EDTA (Invitrogen), and 100ug/ml DNase (Roche). CountBrightTM Absolute Counting Beads, (ThermoFisher) were used as an internal standard according to the manufacturer's instructions to calculate absolute cell counts in cell suspensions.  Antibodies specific for human CD45 (ref 304032, clone HI30), CD3 (317322, OKT3), CD4 (357412, A161A1 / 317440), CD56 (304608, MEM-188), EpCAM (324226/324238, 9C4), CD94 (305520, DX22), KLRB1 (339918, HP-3G10), TIGIT (372716, A15153G), TCR Va24-Ja18 (342922, 6B11), PD-1 (329928, EH12.2H7) were purchased from BioLegend. Antibodies specific for human CD8 (560179, SK1) was purchased from BD Pharmingen. Antibodies specific for human NKG2A (FAB1059P, 131411) and Methothelin (FAB32652P, 420411) were purchased from R&D Systems. Antibodies specific for human CTLA-4 (12-1529-42, 14D3) was purchased from eBioscience. Antibody specific for human NKG2C (130-103-636, REA205) was purchased from Miltenyi.  M5 CAR expression was assessed using biotinylated goat anti-human IgG F(ab’)2 (Jackson ImmunoResearch, 109-066-006) followed by streptavidin (FITC or APC) (BioLegend) or using an anti-idiotype antibody provided by Novartis Pharmaceuticals. Live/dead staining was performed using a Live/Dead Fixable Aqua Dead Cell Stain Kit (Life Technologies following manufacturer’s protocol followed by cell surface staining for 20 minutes at room temperature in the dark. Intracellular staining was performed with the Foxp3/Transcription Factor Staining Buffer set (Thermo Fisher) according to the manufacturer’s instructions. Samples were acquired on an LSRII Fortessa Cytometer (BD Bioscience) and analyzed with FlowJo v10 software (FlowJo, LLC). Sorting assays were performed using a FACS Aria Cytometer (BD Bioscience).
Donors ND566 and ND539: scRNA-seq libraries were generated using a Chromium Single Cell 3’ Library and Gel Bead Kit v3.1 (10x Genomics) according to the manufacturer’s protocol. Briefly, 16,000 CD8+ T cells were sorted by flow cytometry and washed with ice cold PBS + 0.04% BSA. After washing, cells were used to generate single cell gel beads in emulsion. Following reverse transcription, gel beads in emulsion were disrupted and barcoded complementary DNA was isolated and amplified by PCR for 12 cycles. After fragmentation, end repair, and poly A tailing, samples indexes were added and amplified following the manufacturer’s protocol.

treatment_protocol: AsPC-1 cells were routinely seeded in 6-well plates at 1 x 10e6 cells/well the day preceding T cell seeding. M5 CAR T cells (30 - 50% of transduction efficiency) were thawed and rested at 1 x 10e6 cells/ml in T75 flasks with R10 media. After 24 hours, the T cell number (CD45+EpCAM-) was calculated and 2.5 x 10e5 T cells/well were transferred to the AsPC-1 plates. After 3 - 4 days, the cocultures were thoroughly suspended by frequent pipetting and 300 - 400µl of the cell suspension was used for T cell counting assessment and flow cytometry staining. The remaining cell suspension was spun down and the supernatant (conditioned media) was collected and filtered with a 0.45µm filter (Corning). The cells were resuspended in media containing equal amounts of conditioned and fresh R10. The resulting T cell suspension was transferred into AsPC-1-coated plates cells (2.5 x 10e5 T cells/well) for continuous co-culture. This process was repeated for 20-35 days.    Single guide RNA (sgRNA) sequences targeting ID3 and SOX4 were designed using CRISPick sgRNA designer (the Broad Institute [2020]) and Benchling online software (https://www.benchling.com, [2020]) and were synthesized by Integrated DNA Technologies (IDT). Two of five sgRNAs targeting each gene were selected for further experiments after prevalidation. Gene disruption, T cell activation, transduction, expansion, and knockout validation of ID3KO and SOX4KO M5 CAR T cells were performed following an optimized protocol previously described (Agarwal et al., 2021). Since each target locus was defined by two sgRNA cut sites (spanned 100 and 130 bp for SOX4 and ID3 respectively), PCR primers and sequencing primers were designed to detect each target locus. The SpyFI Cas9 protein was purchased from Aldevron and the electroporation enhancer (a non-homologous ssDNA oligonucleotide) was purchased from IDT. LongAmp™ Taq 2X Master Mix (NEB) was used for target sequence amplification and used following manufacturer’s protocol. Analysis of gene editing efficiency was assessed by Sanger sequencing. Synthego’s Performance Analysis ICE (short for Inference of CRISPR Edits) tool, was used to calculate the editing efficiency (https://ice.synthego.com/ [2021]). The following sequences were used for KO generation and editing efficiency validation: Oligonucleotides for genetic deletion of SOX4 and ID3: SOX4 sgRNA #1: 5’-GCTGGTGCAAGACCCCGAGT-3’, SOX4 sgRNA #2: 5’-AGGAGGCGATTCCCAGCTCG-3’, ID3 sgRNAs #1: 5’-TGGCTAAGCTGAGTGCCTCT-3’, ID3 sgRNAs #2 5’-TGGCCAGACTGCGTTCCGAC-3’; Primers for target sequence amplification and sanger sequencing: ID3.PCR.F 5’-ATAAAGAGGCGTGCCTTCCA-3’, ID3.PCR.R 5’- CATCCTTGCCTGGGTGTTCA-3’, ID3.Seq.F 5’-TTCTCTTTGGGGCACCTCTG-3’, ID3.Seq.R 5’-GAAGGTGGGGGCCATCAG-3’; SOX4.PCR.F 5’-CGGAGAACTCCTTCCCCAAATC-3’, SOX4.PCR.R 5’-CTCTTTTTCTGCGCCGGTTTG-3’, SOX4.Seq.F 5’CCGCGAGGGTGTGAGC-3’ and SOX4.Seq.R 5’-TGTAGTCGGGGTAGTCAGCC-3’. To amplify the target sequences on the cDNA samples upon CAE we the SOX4 primers indicated above and the following set of primers for ID3 were used: ID3.cDNAPCR.F 5’- TTGCAGGTCACTGTAGCGG-3’, ID3.cDNAPCR.R 5’- AGGCCACAAGTTCACAGTCC-3’, ID3.cDNASeq.F 5’- TCTTTCTCTTTGGGGCACCTC-3’, ID3.cDNASeq.R 5’- TGGTGAAGTCAAGTGGGCAG-3’. Pos control for cDNA PCR: H3Histone. Human F 5’-AAAGCCGCTCGCAAGAGTGCG-3’ and H3Histone. Human R 5’-ACTTGCCTCCTGCAAAGCAC-3’.

tag: donor

tag: antigen exposure

value: Continuous

tag: genetic background

value: Sox4 KO

tag: exposure time

value: 24 days

value: M5CAR T cells (PBMCs)


title: BM2222_RNA_DMSO_1d_REP2_GTGGCC_S20

tag: cell line

tag: treatment

value: DMSO


title: BM2222_RNA_DMSO_1d_REP1_ACAGTG_S5

tag: timepoint


title: ND566.Day24_Continuous

value: WT


title: ND539.Day20_Continuous

value: 20 days


title: ND539.Day20_Continuous.ID3_KO

value: ID3 KO


title: ND566.Day24_Continuous.ID3_KO


title: ChIA-PET from activated CD4 positive, naive alpha-beta T cell (ENCLB730THG)

status: Public on Nov 04 2021

city: Stanford

first: ENCODE

last: DCC

state: CA

address: 300 Pasteur Dr

institute: ENCODE DCC

description:
https://www.encodeproject.org/experiments/ENCSR402NAQ/
***************
biological replicate number: 2
technical replicate number: 1
description: RNAPII ChIA-PET in Naive_CD4_Activated_Day2
experiment encode accession: ENCSR402NAQ
assay title: ChIA-PET
assembly: GRCh38
encode release date: 2021-07-21
lab: Yijun Ruan, JAX
library encode accession: ENCLB730THG
fragmentation methods: sonication (Branson Sonifier 250)
size range: 350-600
instrument models: Illumina MiSeq,Illumina NovaSeq 6000

data_processing: See GSM*_README.txt supplementary file linked below

source_name: Homo sapiens male adult (43 years) activated CD4 positive, naive alpha-beta T primary cell treated with anti-CD3 and anti-CD28 coated beads

tag: antibody

value: primary cell

tag: biosample encode accession

tag: Sex

value: male

tag: dev stage

value: adult

tag: age

value: 43 year

tag: lab

value: Yijun Ruan, JAX

tag: source

value: AllCells

tag: treatments

value: treatment_term_name:anti-CD3 and anti-CD28 coated beads; treatment_type:antibody;

tag: link

value: ENCBS753APS at ENCODE; https://www.encodeproject.org/ENCBS753APS/

value: Derived from ENCODE donor ENCDO791UJG; https://www.encodeproject.org/ENCDO791UJG/


title: ChIA-PET from activated CD4-positive, alpha-beta T cell (ENCLB372VVA)

description:
https://www.encodeproject.org/experiments/ENCSR314TNQ/
***************
biological replicate number: 1
technical replicate number: 1
description: RNAPII ChIA-PET in CD4_Activated_Day2
experiment encode accession: ENCSR314TNQ
assay title: ChIA-PET
assembly: GRCh38
encode release date: 2021-07-21
lab: Yijun Ruan, JAX
library encode accession: ENCLB372VVA
fragmentation methods: sonication (Branson Sonifier 250)
size range: 350-600

source_name: Homo sapiens male adult (20 years) activated CD4-positive, alpha-beta T primary cell treated with anti-CD3 and anti-CD28 coated beads

value: 20 year

value: ENCBS840LNC at ENCODE; https://www.encodeproject.org/ENCBS840LNC/

value: Derived from ENCODE donor ENCDO394DKX; https://www.encodeproject.org/ENCDO394DKX/


title: ChIA-PET from activated CD4 positive, naive alpha-beta T cell (ENCLB842UTD)

description:
https://www.encodeproject.org/experiments/ENCSR731OFF/
***************
biological replicate number: 2
technical replicate number: 1
description: CTCF ChIA-PET in Naive_CD4_Activated_Day2
experiment encode accession: ENCSR731OFF
assay title: ChIA-PET
assembly: GRCh38
encode release date: 2021-07-21
lab: Yijun Ruan, JAX
library encode accession: ENCLB842UTD
fragmentation methods: sonication (Branson Sonifier 250)
size range: 350-600

value: CTCF


title: ChIA-PET from activated CD4-positive, alpha-beta T cell (ENCLB714GLI)

description:
https://www.encodeproject.org/experiments/ENCSR733XLQ/
***************
biological replicate number: 1
technical replicate number: 1
description: RNAPII ChIA-PET in CD4_Activated_Day7
experiment encode accession: ENCSR733XLQ
assay title: ChIA-PET
assembly: GRCh38
encode release date: 2021-07-21
lab: Yijun Ruan, JAX
library encode accession: ENCLB714GLI
fragmentation methods: sonication (Branson Sonifier 250)
size range: 350-600

source_name: Homo sapiens male adult (20 years) activated CD4-positive, alpha-beta T primary cell treated with 10 ng/mL Interleukin-2 , anti-CD3 and anti-CD28 coated beads

value: treatment_term_id:UniProtKB:P60568; treatment_term_name:Interleukin-2; treatment_type:protein; treatment_term_name:anti-CD3 and anti-CD28 coated beads; treatment_type:antibody;

value: ENCBS718HAG at ENCODE; https://www.encodeproject.org/ENCBS718HAG/


title: ChIA-PET from activated CD4-positive, alpha-beta T cell (ENCLB717ULN)

description:
https://www.encodeproject.org/experiments/ENCSR733XLQ/
***************
biological replicate number: 2
technical replicate number: 1
description: RNAPII ChIA-PET in CD4_Activated_Day7
experiment encode accession: ENCSR733XLQ
assay title: ChIA-PET
assembly: GRCh38
encode release date: 2021-07-21
lab: Yijun Ruan, JAX
library encode accession: ENCLB717ULN
fragmentation methods: sonication (Branson Sonifier 250)
size range: 350-600

value: ENCBS860YXJ at ENCODE; https://www.encodeproject.org/ENCBS860YXJ/


title: ChIA-PET from activated CD4 positive, naive alpha-beta T cell (ENCLB264OYK)

description:
https://www.encodeproject.org/experiments/ENCSR446MTI/
***************
biological replicate number: 2
technical replicate number: 1
description: RNAPII ChIA-PET in Naive_CD4_Activated_Day7
experiment encode accession: ENCSR446MTI
assay title: ChIA-PET
assembly: GRCh38
encode release date: 2021-07-21
lab: Yijun Ruan, JAX
library encode accession: ENCLB264OYK
fragmentation methods: sonication (Branson Sonifier 250)
size range: 350-600
instrument models: Illumina MiSeq,Illumina NovaSeq 6000

source_name: Homo sapiens male adult (43 years) activated CD4 positive, naive alpha-beta T primary cell treated with 10 ng/mL Interleukin-2 , anti-CD3 and anti-CD28 coated beads

value: ENCBS338LVO at ENCODE; https://www.encodeproject.org/ENCBS338LVO/


title: Ly6Chigh blood circulating monocytes rep3 (re-analysis)

status: Public on Nov 16 2021

first: Laszlo

last: Halasz

state: NY

address: 1470 Madison Avenue

department: Department of Oncological Sciences

institute: Mount Sinai Icahn School of Medicine

data_processing:
RNA-seq: Raw reads were aligned to the mm10 (GRCm38) genome with STAR using nf-core/rnaseq pipeline version 3.2. Genes counts were quantified with Salmon using default parameters.
Genome_build: mm10
Supplementary_files_format_and_content: Raw and normalized count matrices.

source_name: Ly6Chigh blood circulating monocytes

extract_protocol:
Total RNA was isolated with TRIZOL reagent according to the manufacturer’s recommendation. 20ug glycogen (Ambion) was added as carrier for RNA ethanol precipitation
cDNA library for RNA-Seq was generated from 100ng-400ng total RNA using TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly poly-A tailed RNA molecules were pulled down with poly-T oligo attached magnetic beads. Following purification, mRNA was fragmented with divalent cations at 85C, and then cDNA was generated by random primers and SuperScript II enzyme (Life Technologies). Second strand synthesis was performed followed by end repair, single `A` base addition and ligation of barcode indexed adaptors to the DNA fragments. Adapter specific PCRs were performed to generate sequencing libraries. Libraries were size selected with E-Gel EX 2% agarose gels (Life Technologies) and purified by QIAquick Gel Extraction Kit (Qiagen). Libraries were sequenced on HiSeq 2500 instrument.

tag: tissue

value: Blood

tag: strain

value: female

value: adult 2-4 months old

tag: cell type

value: Ly6Chigh blood circulating monocytes


title: TA muscle Ly6Clow macrophage day 2 after CTX rep 3 (re-analysis)

source_name: Ly6Clow macrophages day 2 after CTX

value: TA muscle

value: male

value: Ly6Clow macrophages

value: CTX

tag: time

value: day 2 after CTX


title: TA muscle Ly6Chigh macrophage day 2 after CTX rep 2 (re-analysis)

source_name: Ly6Chigh macrophages day 2 after CTX

value: Ly6Chigh macrophages


title: TA muscle macrophage day 4 after CTX rep 2 (re-analysis)

source_name: Ly6Clow macrophages day 4 after CTX

value: day 4 after CTX


title: TA muscle Ly6Chigh macrophage day 1 after CTX rep 2 (re-analysis)

source_name: Ly6Chigh macrophages day 1 after CTX

value: day 1 after CTX


title: TA muscle Ly6Chigh macrophage day 2 after CTX rep 1 (re-analysis)


title: TA muscle macrophage day 4 after CTX rep 3


title: TA muscle Ly6Chigh macrophage day 1 after CTX rep 1 (re-analysis)


title: TA muscle Ly6Clow macrophage day 2 after CTX rep 1 (re-analysis)


title: TA muscle Ly6Clow macrophage day 2 after CTX rep 2 (re-analysis)


title: Ly6Chigh blood circulating monocytes rep1 (re-analysis)


title: Ly6Chigh blood circulating monocytes rep2 (re-analysis)


title: TA muscle Ly6Chigh macrophage day 2 after CTX rep 3 (re-analysis)


title: TA muscle macrophage day 4 after CTX rep 4


title: TA muscle macrophage day 4 after CTX rep 1 (re-analysis)


title: TA muscle Ly6Chigh macrophage day 1 after CTX rep 3 (re-analysis)