title: vWAT_mac_4OHE1_rep2

data_processing:
Unmanipulated reads were aligned to the mouse geneome (mm9) using STAR, producing SAM files.
SAM files were converted to tag directories using HOMER.
For reporting, HOMER was used to calculate nomalized reads per gene (reads per kilobase per million mapped reads, or RPKM).
For differential expression analysis, HOMER was used to count raw reads per gene. The raw read counts were used as input to DESeq2 or EdgeR to call differential expression.
Genome_build: mm9
Supplementary_files_format_and_content: three xlsx spreadsheets containing: 1) RPKM values for each gene 2), 3) differential expression analysis outputs

source_name: F480+ CD11b+ vWAT macrophage

growth_protocol: Visceral white adipose tissue (vWAT) was isolated from mice (see below) and stromal vascular fraction (SVF) prepared by mincing tissue and incubating in DMEM with 0.1% collagenase (Sigma C6885) and 5% BSA for 1 hour at room temperature with gentle shaking. Digested tissue was filtered through a 70mm filter, RBCs lysed, and SVF stained on ice in FACs buffer (PBS with 10mM HEPES and 5% BSA) using standard techniques. Briefly, after incubation in Fc Block (BD Biosciences), cells were stained with anti-CD45-PerCP-Cy5.5 (clone 30-F11, BioLegend), anti-F4/80-PE (clone BM8, eBioscience), and anti-CD11b-APC (clone M1/70, eBioscience). After washes, cells were resuspended in FACs buffer with DAPI (Thermo Fisher D1306, reconstituted according to manufacturer instructions, used at 1:100,000) for exclusion of dead cells and F4/80+ CD11b+ vWAT macrophages were sorted.

extract_protocol:
vWAT macrophages were sorted directly into Trizol-LS (Invitrogen) and total RNA isolated according to manufacturer’s instructions using chloroform extraction and glycogen-assisted RNA precipitation with GlycoBlue (Ambion).
mRNA was isolated from total RNA and converted into sequencing libraries using mRNA HyperPrep kit (KAPA/Roche) and Illumina-compatible NEXTFlex sequencing adaptors (Bioo) according to manufacturer’s instructions. Libraries were PCR amplified, quantified with Illumina Library qPCR quantification kit (KAPA/Roche), and pooled for sequencing on HiSeq2500 (Illumina).

treatment_protocol: vWAT macrophages were sorted from 12-week-old male C57BL/6N mice (Charles River) fed a high-fat diet (HFD, Research Diets Inc, D12492, 60 kcal% fat) and given subcutaneous (SQ) EtOH vehicle control or estrogen injections (10mg/kg) every 6 days in rear flank for a total of 30 days.

value: male

tag: treament

value: F480+ CD11b+ vWAT macrophage


title: 651_NT_H3K27ac_Chip-seq

status: Public on Apr 04 2021

type: SRA

city: Hong Kong

first: Alfred Sze-Lok

last: Cheng

address: Rm 405A, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, CUHK

department: School of Biomedical Sciences

country: Hong Kong

institute: The Chinese University of Hong Kong

library_source: genomic

data_processing:
bowtie2 v2.2.9 for alignment
Reads were trimmed 10-bp from the front and the back to produce 80-bp. Only reads with mapQ >10 and with duplicates removed by rmdup were used for subsequent analysis.
MACS2 v2.1.0 for peak calling
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files (fold change over control or signal p-value) were generated from bedGraph files with UCSC tools (bedClip, bed-Graph-ToBigWig)

molecule: genomic DNA

organism: Homo sapiens

source_name: Adjacent non-tumor liver tissue from NAFLD-HCC patient

extract_protocol:
Tissues were directly crosslinked with 1% formaldehyde and the reaction was stopped by glycine at room temperature (RT). Fixed tissues were lysed with SDS-Lysis buffer containing proteinase inhibitor. The lysates were fragmented into 200-300bp by Bioruptor (Diagenode, Denville, New Jersey, USA).
ChIP-graded anti-H3K27ac antibody (39133, Active Motif, 1:500) or control normal Rabbit IgG (2729, Cell Signaling Technology, 1:500) were conjugated with magnetic beads (Magna ChIP™ Protein A+G Magnetic Beads, Life Technologies) by rotation in cold room (4°C) for 1 hour. Meanwhile, the cell lysates were incubated with magnetic beads and rotated in cold room (4°C) for 1 hour, to remove endogenous antibodies. Cell lysates were then incubated with magnetic beads-conjugated antibodies and rotated in cold room (4°C) for 12-16 hours. After washing and reversed crosslinking, the immunoprecipitated DNA were purified by purification kit (Qiagen). Whole-genome amplification was performed using the WGA4 kit (Sigma-Aldrich) and BpmI-WGA primers and digested with BpmI (New England Biolab) before the NEBNext ChIP-seq library prep reagent set (New England Biolab). Each library was sequenced to an average depth of 20 to 30 million raw reads on HiSeq4000 using 100-bp pair-end reads.

treatment_protocol: HCC patients with a clinical background of no HBV and/or HCV infection, no history of alcoholic liver who underwent hepatectomy at the Prince of Wales Hospital (Hong Kong) were included in this study. The specimens were processed immediately after surgery and snap-frozen in liquid nitrogen for epigenomic analyses.

tag: tissue

value: Adjacent non-tumor liver tissue


title: vWAT_mac_4OHE1_rep1

city: Berkeley

first: Greg

last: Timblin

state: CA

department: MCB

country: USA

institute: UC Berkeley

library_source: transcriptomic

molecule: total RNA

organism: Mus musculus

tag: strain

tag: Sex

tag: cell type


title: vWAT_mac_E2_rep2


title: 768_NT_RNA-seq

data_processing:
tophat v2.0.13 for alignment
Cufflinks v2.2.1 for abundance quantification and differential expression
Genome_build: hg19
Supplementary_files_format_and_content: txt files containing abundance quantification (count & FPKM) & differential expression results

extract_protocol:
Total RNA was extracted from frozen tissues using Trizol (Invitrogen) and purified on TNeasy MinElute columns (QIAGEN). RNA integrity was confirmed on the Bioanalyzer (Agilent Technologies, Palo Alto, CA).
Library preparation and sequencing were performed by BGI Tech (Hong Kong) using Illumina HiSeq standard protocols and paired-end (101-bp) sequencing on HiSeq4000 sequencer (Illumina).

treatment_protocol: HCC patients with a clinical background of no HBV and/or HCV infection, no history of alcoholic liver who underwent hepatectomy at the Prince of Wales Hospital (Hong Kong) were included in this study. The specimens were processed immediately after surgery and snap-frozen in liquid nitrogen for transcriptomic analyses.


title: 768_T_RNA-seq

source_name: Tumor tissue from NAFLD-HCC patient

tag: ajcc stage

value: AJCC 3

value: Tumor tissue


title: vWAT_mac_EtOH_rep2

value: EtOH


title: 741_NT_RNA-seq


title: 705_T_RNA-seq


title: 741_T_RNA-seq


title: BMDM_ctrl1_EtOH

data_processing:
Unmanipulated reads were aligned to the mouse geneome (mm9) using STAR, producing SAM files.
SAM files were converted to tag directories using HOMER.
For reporting, HOMER was used to calculate nomalized reads per gene (reads per kilobase per million mapped reads, or RPKM).
For differential expression analysis, HOMER was used to count raw reads per gene. The raw read counts were used as input to DESeq2 or EdgeR to call differential expression.
Genome_build: mm9
Supplementary_files_format_and_content: two xlsx spreadsheet containing: 1) RPKM values for each gene 2) differential expression analysis output

source_name: bone marrow-derived macrophage

growth_protocol: Bone marrow-derived macrophages (BMDMs) were prepared by flushing leg bones from 8-week-old female C57BL/6N mice (Charles River), filtering cells through a 40mm filter, RBC lysis, and plating in phenol red-free DMEM (Corning 17-205-CV) supplemented with 10% charcoal-stripped FBS (HyClone SH30068.03), pen/strep (Gibco), and 20ng/mL murine M-CSF (Shenandoah) in 15cm non-TC treated Petri dishes. Media was refreshed every 3 days. On day 7, cells were harvested by scraping and plated in TC-treated 24 well plates at 7.5x104 cells per well in 1mL of media.

extract_protocol:
Cells were directly lysed in 300mL Trizol (Invitrogen) and total RNA isolated with Direct-zol RNA Miniprep kit (Zymo).
mRNA was isolated from total RNA and converted into sequencing libraries using mRNA HyperPrep kit (KAPA/Roche) and Illumina-compatible NEXTFlex sequencing adaptors (Bioo) according to manufacturer’s instructions. Libraries were PCR amplified, quantified with Illumina Library qPCR quantification kit (KAPA/Roche), and pooled for sequencing on HiSeq2500 (Illumina).

treatment_protocol: Cells were treated with EtOH vehicle control (1:1000) or estrogens (1mM stocks in EtOH diluted 1:1000 for 1mm final concentration) for 1 hour, followed by 6 hour LPS stimulation (100ng/mL).

value: female

tag: treatment

value: hydroxyestrogen

value: bone marrow-derived macrophage


title: vWAT_mac_E2_rep1


title: vWAT_mac_EtOH_rep1


title: 705_NT_RNA-seq


title: 796_NT_RNA-seq


title: BMDM_ctrl2_E2


title: 796_T_RNA-seq

value: AJCC 1


title: BMDM_ctrl3_16Epi


title: 717_T_RNA-seq


title: BMDM_hydroxyestrogen2_4OHE1

value: control


title: 754_T_RNA-seq


title: BMDM_hydroxyestrogen1_2OHE2


title: BMDM_hydroxyestrogen3_2OHE1

value: bone marrow-derived macrophage (BMDM)


title: 717_NT_RNA-seq


title: Refed MED13-LKO with Fructose Rep2

city: Bronx

first: Jeffrey

middle: E

last: Pessin

state: NY

address: 1301 Morris Park Avenue

department: Medicine and Molecular Pharmacology

institute: Albert Einstein College of Medicine

data_processing:
Sequencing reads were trimmed using trim galore v0.6.5 with option --stringency 5
Transcript-level counts were estimated by Kallisto v0.46.0
Differential expression analysis was performed with DESeq2 v1.24.0
Genome_build: mm10
Supplementary_files_format_and_content: csv file containing normalized counts for each gene and each sample, output from DESeq2

source_name: Liver

extract_protocol:
Total RNA was isolated from tissues by using RNeasy kit from Qiagen according to manufature's protocol.
The total RNA was sent to Novogene Corporation Inc for library construction and sequencing. Company's strandard protocol include in generating 150 bp cDNA fragments, paired and two-ended reads, and 30 million fragments/library construction for Illumina machine.

treatment_protocol: Normal Chow: lab diet #5053

tag: genotype/variation

value: MED13 knockout (MED13-LKO)

tag: mice age

value: 12 weeks

tag: diet

value: Refed 4 hrs with normal chow and 30 % fructose water


title: 715_T_RNA-seq


title: KO-MCD rep4

city: Los Angeles

first: Lu

last: Li

address: Room 706, 1985 Zonal Ave

department: School of Pharmacy

institute: University of Southern California

description: Sample 16

data_processing:
The reads were mapped to the latest UCSC transcript set using Bowtie2 version 2.1.0 and the gene expression level was estimated using RSEM v1.2.15.
TMM (trimmed mean of M-values) was used to normalize the gene expression.  Mapping statistics were above average; between 62.92% and 76.70% of reads mapped to the transcriptome for any given sample.
Genome_build: rn6
Supplementary_files_format_and_content: txt files were generated; Scores in Rsem_count.txt represent the counts of genes and isoforms; Scores in TMM_normalized_reads_count_per_million.txt represent the normalized gene expression data by TMM method for each sample

organism: Rattus norvegicus

source_name: liver

growth_protocol: The TSPO WT and KO rats were fed normally during the first 12 weeks of postnatal period

extract_protocol:
Total RNA was harvested from the liver and prepared using Ambion® RiboPure™ Kit (Thermofisher Scientific, AM1924), according to the manufacture's instructions.
Libraries for RNA-Seq were prepared with Kapa Stranded mRNA. The workflow consisted of mRNA enrichment, cDNA generation, and end repair to generate blunt ends, A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane.

treatment_protocol: The 12 weeks old TSPO WT and KO rats were fed with chow and MCD, respectively, for 8 weeks.

value: Sprague Dawley

tag: age

value: post natal 20 weeks

value: Liver

tag: genotype

value: tspo -/-

tag: feeding

value: methionine-choline deficient


title: WT-MCD rep4

description: Sample 15

value: wild type


title: KO-CHOW rep4

description: Sample 14

value: chow


title: Refed MED13-LKO with Fructose Rep1


title: Refed MED13-LKO with Sucrose Rep2

value: Refed 4 hrs with normal chow and 30 % sucrose water


title: WT-CHOW rep4

description: Sample 13


title: 715_NT_RNA-seq

value: Adjacent non-tumor liver tissue


title: 754_NT_RNA-seq


title: KO-MCD rep3

description: Sample 12


title: Refed MED13-LKO with Sucrose Rep1


title: 707_T_RNA-seq

value: AJCC 3

value: Tumor tissue


title: KO-MCD rep2

description: Sample 11


title: Fasted MED13-LKO Rep2

value: Fasted overnight 16 hrs


title: 678_T_RNA-seq

value: AJCC 1


title: Fasted MED13-LKO Rep1


title: KO-MCD rep1

description: Sample 10


title: WT-MCD rep3

description: Sample 9


title: WT-MCD rep1

description: Sample 7


title: WT-MCD rep2

description: Sample 8


title: Refed Control with Fructose Rep1

value: control (Med13fl/fl)


title: MLXIP-KO, rep3

type: RNA

city: Munich

first: Maxim

last: Barenboim

address: Koelner Platz 1

department: Department of Pediatrics

country: Germany

institute: TECHNICAL UNIVERSITY OF MUNICH

hyb_protocol: cRNA was hybridized to Affymetrix Human Gene 1.0 ST arrays.

scan_protocol: GeneChip Scanner 3000 7G System, AGCC software (Affymetrix).

data_processing: RMA normalization using Expression Console software (Build 1.1; Affymetrix).

label: biotin

label_protocol: Total RNA (200 ng) was amplified and labeled using Affymetrix GeneChip Whole Transcript Sense Target Labeling Kit.

growth_protocol: Cell lines were grown in plastic cell culture flasks in a humidified incubator at 37°C in 5% CO2 atmosphere in RPMI 1640 media containing 10% heat-inactivated fetal bovine serum, 1% glutamine, 100 µg/mL gentamycin and 200 mg/dl D-glucose.

extract_protocol: RNA was isolated using TRIZOL according to the manufacturer's instructions.

treatment_protocol: Oligonucleotides cloned into lentiviral vector LentiGuide-Puro. Lentiviral constructs LentiCas9-Blast and LentiGuide-Puro plasmid with ligated selected sgRNA transfected into HEK293T. Viral supernatants isolated 48h after transfection and filtered. Nalm6 cells transduced with viral supernatant. Blasticidin-resistant cells transduced with viral supernatant containing LentiGuide-Puro with ligated selected sgRNA.

value: B cell precursor leukemia

tag: cell line

value: Human Pre-B Cell Line Nalm6

value: MLXIP-KO


title: 651_NT_RNA-seq


title: MLXIP-WT, rep2

value: MLXIP-WT


title: MLXIP-KO, rep1


title: Refed Control with Fructose Rep2

value: 12 weeks

value: Refed 4 hrs with normal chow and 30 % fructose water


title: Refed Control with Sucrose Rep1


title: KO-CHOW rep3

description: Sample 6

value: Sprague Dawley

value: post natal 20 weeks

value: Liver

value: tspo -/-


title: cytotrophoblast cells-IL-11+IL-6-6h-rep3

city: Tampa

first: Asli

last: Ozmen

state: Florida

address: 560 Channelside Drive

department: ObGyn

institute: University of South Florida

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-11 and 10ng/ml rhIL-6 for 6 h rep 3

hyb_protocol: Amplified RNA was hybridized to HumanHT-12 v4 Expression BeadChip using Illumina's Whole-Genome Gene Expression Direct Hybridization Assay Guide.

scan_protocol: BeadChips were scanned using the Illumina HiScan System

data_processing: GeneSpring GX12.5 software (Agilent Technologies, Redwood City, CA) were used to analyze raw data without normalization. Gene readouts were normalized to the 75th percentile of the distribution of all measurements in each chip. Normalization for each gene across chips was performed using the median value of each gene throughout different chips in the same experimental condition.

source_name: Primary cytotrophoblast cells isolated from human term placenta treated with IL-11+ IL-6 for 6h

label_protocol: Total RNA samples were normalized to 500ng using Thermo Scientific NanoDrop concentration readings. Samples were then amplified using Ambion's Illumina TotalPrep RNA Amplification Kit (P/N AMIL1791) following Ambion's protocol. Amplified RNA samples were measured for their concentration using the Thermoscientific NanoDrop and normalized to 750ng.

growth_protocol: Cytotrophoblasts (CTBs) isolated from term placental specimens obtained from cesarean delivery without labor. Briefly, after removal of decidual tissues, fragments of chorionic villi were subjected to enzymatic digestion in a Hanks’ Balanced Salt Solution (Invitrogen, Carlsbad, CA) solution containing 0.25% trypsin (Invitrogen), 0.2% DNase I (Roche, Indianapolis, IN), 25 mM HEPES, 2 mM CaCl2 and 0.8 mM MgSO4 in at 37°C for 90 min. Cell suspension were then separated using a discontinuous (30, 35, 45, 50%) Percoll gradient (GE Healthcare, Piscataway, NJ). Cells in 35-45% Percoll interface were collected and immune-purified using mouse anti-human CD9 (R&D Inc., Minneapolis, MN) and mouse anti-CD45 (R&D) antibodies for negative selection by removing fibroblasts and immune cells. Then, the unbound cells were collected, washed, and cultured in 6-well plates in DMEM/F12 (Gibco, Life Technologies Corporation, Grand Island, NY) containing 10% FBS (Gemini Bio Products, West Sacramento, CA).

extract_protocol: Total RNA was isolated using Qiagen miRNeasy Mini Kit (Qiagen, Valencia, CA) and RNeasy MinElute Cleanup Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions.

treatment_protocol: Following 24 h incubation in serum-free media, CTBs were treated with vehicle or recombinant human IL-11 (10 ng/ml, R&D) or IL-6 (10 ng/ml, R&D) or IL-11+IL-6 for 6 h for microarray analysis.

value: villous tissue obtained from human term placenta

tag: cell

value: cultured cytotrophoblast cells

tag: gender

value: female

tag: gestational stage

value: term un-complicated pregnancy

value: rhIL-11+ rhIL-6 (10ng/ml each) for 6h


title: MLXIP-KO, rep2


title: KO-CHOW rep2

description: Sample 5

value: chow


title: 707_NT_RNA-seq

value: Adjacent non-tumor liver tissue


title: Fasted Control Rep1

value: control (Med13fl/fl)

value: Fasted overnight 16 hrs


title: UV-2

city: Cambridge

first: Elaina

last: Thomas

state: MA

address: 15 Vassar St

department: Civil and Environmental Engineering

institute: MIT

description:
Prochlorococcus strain MIT0604
UV_treatment_vs_control_sense_ThomasGenomeGFF_notFilteredForSig_sameGenesGroupedTogether.csv
Mitomycin_C_treatment_vs_UV_treatment_sense_ThomasGenomeGFF_notFilteredForSig_sameGenesGroupedTogether.csv
UV_treatment_vs_control_antisense_ThomasGenomeGFF_notJustSig_groupingDuplicateGenes.csv
Mitomycin_C_treatment_vs_UV_treatment_antisense_ThomasGenomeGFF_notJustSig_groupingDuplicateGenes.csv

data_processing:
Adapter trim raw reads; software: bbduk v38.16, parameters: ktrim=r k=23 mink=11 hdist=1
Quality trim adapter trimmed reads; software: bbduk v38.16, parameters: qtrim=rl, trimq=6
Map quality trimmed reads to genome; software: Burrows-Wheeler Aligner v0.7.16a-r1181; parameters: BWA-backtrack algorithm, mapped to Prochlorococcus strain MIT0604 reference genome (available from https://github.com/thackl/pro-tycheposons/)
Count reads that mapped to each feature; software: HTSeq v0.11.2; parameters: --type=CDS --idattr=ID -a 0 --nonunique all
Differential expression analysis; software: DESeq2 R package v1.24.0; parameters: standard DESeq2 functions and workflow. Differential expression tests were performed on mitomycin C vs. control and UV shock vs. control comparisons with the Wald test, using a negative binomial generalised linear model. P-values were corrected for multiple testing with the Benjamini–Hochberg procedure. As was suggested by the DESeq2 authors(Love et al. 2014), genes with an adjusted p-value of <0.1 were considered to have significantly different expression between a given pair of treatments.
Genome_build: Reference genome used for read alignment: Prochlorococcus strain MIT0604 genome (available from https://github.com/thackl/pro-tycheposons/)
Supplementary_files_format_and_content: HTSeq raw counts (sense orientation): text file of each gene and the number of reads determined to align to the given gene in the sense orientation. Gene counts were determined using HTSeq.
Supplementary_files_format_and_content: HTSeq raw counts (antisense orientation): text file of each gene and the number of reads determined to align to the given gene in the antisense orientation. Gene counts were determined using HTSeq.
Supplementary_files_format_and_content: DeSeq2 differential expression results (including insignificant log2fold changes) (sense orientation): Results of  differential expression analysis (using DeSeq2) on gene expression in the sense orientation. For each gene, we include the average of normalized count values, dividing by size factors, taken over all samples (baseMean), the effect size estimate of the gene expression change between the comparison and control groups (log2FoldChange), the standard error estimate for the log2 fold change estimate (lfcSE), the value of the test statistic for the gene (stat), the p-value of the test for the gene (pvalue), and the adjusted p-value for multiple testing for the gene (padj). Genes with an adjusted p-value of <0.1 were considered to have significantly different expression between a given pair of treatments.
Supplementary_files_format_and_content: DeSeq2 differential expression results (including insignificant log2fold changes) (antisense orientation): Results of differential expression analysis (using DeSeq2) on gene expression in the antisense orientation. For each gene, we include the average of normalized count values, dividing by size factors, taken over all samples (baseMean), the effect size estimate of the gene expression change between the comparison and control groups (log2FoldChange), the standard error estimate for the log2 fold change estimate (lfcSE), the value of the test statistic for the gene (stat), the p-value of the test for the gene (pvalue), and the adjusted p-value for multiple testing for the gene (padj). Genes with an adjusted p-value of <0.1 were considered to have significantly different expression between a given pair of treatments.

organism: Prochlorococcus sp. MIT 0604

source_name: Bacterial culture

growth_protocol: Prochlorococcus MIT0604 strain were grown to exponential phase.

extract_protocol:
Cells were harvested by centrifugation at 12,000 g for 12 min, 20°C and RNA was extracted using the mirVana microRNA (miRNA) extraction kit (Ambion, Carlsbad, CA, USA)
Libraries were constructed using the KAPA RNA HyperPrep kit (Illumina) and used the RiboZero kit (Illumina) for ribosomal RNA depletion

treatment_protocol: 3 cultures were treated with mitomycin C at a final concentration of 15 µg mL-1 for 2 hours; 3 cultures were applied a UV shock by irradiating cells at room temperature for 30 s at 254 nm (UV 100 µW cm-2); the 3 remaining cultures were kept as control with no treatment.

value: Treated with a UV shock by irradiating cells at room temperature for 30 s at 254 nm (UV 100 µW cm-2)


title: 678_NT_RNA-seq


title: cytotrophoblast cells-IL-11+IL-6-6h-rep1

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-11 and 10ng/ml rhIL-6 for 6 h rep 1


title: 651_T_RNA-seq

value: AJCC 1

value: Tumor tissue


title: KO-CHOW rep1

description: Sample 4


title: cytotrophoblast cells-IL-6-6h-rep2

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-6 for 6 h rep 2

source_name: Primary cytotrophoblast cells isolated from human term placenta treated with IL-6 for 6h

value: rhIL-6 (10ng/ml) for 6h


title: Refed Control with Sucrose Rep2

value: 12 weeks

value: Refed 4 hrs with normal chow and 30 % sucrose water


title: MLXIP-WT, rep1

value: B cell precursor leukemia

value: Human Pre-B Cell Line Nalm6

value: MLXIP-WT


title: WT-CHOW rep3

description: Sample 3

value: Sprague Dawley

value: post natal 20 weeks

value: Liver

value: wild type


title: cytotrophoblast cells-IL-6-6h-rep1

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-6 for 6 h rep 1

value: villous tissue obtained from human term placenta

value: cultured cytotrophoblast cells

value: female

value: term un-complicated pregnancy


title: Fasted Control Rep2

value: control (Med13fl/fl)

value: Fasted overnight 16 hrs


title: UV-1

value: Treated with a UV shock by irradiating cells at room temperature for 30 s at 254 nm (UV 100 µW cm-2)


title: UV-3


title: WT-CHOW rep2

description: Sample 2

value: chow


title: cytotrophoblast cells-IL-11+IL-6-6h-rep2

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-11 and 10ng/ml rhIL-6 for 6 h rep 2

value: rhIL-11+ rhIL-6 (10ng/ml each) for 6h


title: WT-CHOW rep1

description: Sample 1


title: cytotrophoblast cells-IL-11-6h-rep3

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-11 for 6 h rep 3

source_name: Primary cytotrophoblast cells isolated from human term placenta treated with IL-11 for 6h

value: rhIL-11 (10ng/ml) for 6h


title: MMC-3

description:
Prochlorococcus strain MIT0604
Mitomycin_C_treatment_vs_control_sense_ThomasGenomeGFF_notFilteredForSig_sameGenesGroupedTogether.csv
Mitomycin_C_treatment_vs_UV_treatment_sense_ThomasGenomeGFF_notFilteredForSig_sameGenesGroupedTogether.csv
Mitomycin_C_treatment_vs_control_antisense_ThomasGenomeGFF_notJustSig_groupingDuplicateGenes.csv
Mitomycin_C_treatment_vs_UV_treatment_antisense_ThomasGenomeGFF_notJustSig_groupingDuplicateGenes.csv

value: treated with mitomycin C at a final concentration of 15 µg mL-1 for 2 hours


title: tumor tissue sample 1272

city: Guangzhou

first: Xin

last: Chen

address: 651 Dongfeng dong Road

country: China

institute: Sun Yat-sen University Cancer Center

description:
TR1272
circularRNA expression in the tissue

hyb_protocol: The labeled cRNAs were hybridized onto the Arraystar Human circRNA Array (8x15K, Arraystar)

scan_protocol: After having washed the slides, the arrays were scanned by the Agilent Scanner G2505C

data_processing:
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images.
Quantile normalization and subsequent data processing were performed using the R software package. Differentially expressed circRNAs with statistical significance between two groups were identified through Volcano Plot filtering. Differentially expressed circRNAs between two samples were identified through Fold Change filtering.

source_name: Homo sapiens

label_protocol: Total RNA from each sample was treated with Rnase R to enrich circular RNA. The enriched circular RNA was then amplified and transcribed into fluorescent cRNA utilizing ramdom primer according to Arraystar Super RNA Labeling protocol (Arraystar, Inc.).

extract_protocol: Trizol Extraction

treatment_protocol: Total RNAs were digested with Rnase R (Epicentre, Inc.) to remove linear RNAs and enrich circular RNAs

tag: disease state

value: urothelial carcinoma of the bladder

value: tumor tissue


title: tumor tissue sample 1268

description:
TR1268
circularRNA expression in the tissue

value: male

tag: tissue


title: MMC-1


title: MMC-2


title: tumor tissue sample 1255

description:
TR1255
circularRNA expression in the tissue


title: cytotrophoblast cells-IL-6-6h-rep3

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-6 for 6 h rep 3

value: rhIL-6 (10ng/ml) for 6h


title: cytotrophoblast cells-IL-11-6h-rep2

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-11 for 6 h rep 2


title: Control-3

description:
Prochlorococcus strain MIT0604
Mitomycin_C_treatment_vs_control_sense_ThomasGenomeGFF_notFilteredForSig_sameGenesGroupedTogether.csv
UV_treatment_vs_control_sense_ThomasGenomeGFF_notFilteredForSig_sameGenesGroupedTogether.csv
Mitomycin_C_treatment_vs_control_antisense_ThomasGenomeGFF_notJustSig_groupingDuplicateGenes.csv
UV_treatment_vs_control_antisense_ThomasGenomeGFF_notJustSig_groupingDuplicateGenes.csv

value: untreated


title: WT2_S3

city: Shanghai

first: Jiahui

last: Du

address: Zhizaoju Road 639

institute: Shanghai Jiao Tong University

description: We examined chromatin structure using ATAC-seq

data_processing:
Library sequencing quality was assessed using FastQC v0.11.8 and trimmed with Cutadapt v1.18 with default parameters
Trimmed libraries were aligned to the mm10 mouse genome using bowtie2 v2.3.5
All unique alignments (MAPQ>=10) were further filtered by Picard MarkDuplicates v2.21.4 to remove duplicates
ATAC-Seq peaks were called by MACS2  v2.1.1 (callpeak -g ‘mm’, --format BAMPE) for each library with a stringent statistical criterion (fold enrichment > 4, p-value < 1e-5 and FDR < 0.05).
These peaks were merged using Bedtools into 13500 chromatin accessibility regions of interest. Raw read counts from the three libraries on these regions were used as input for edgeR v3.20.7.2 to identify differential ATAC-Seq peaks between WT and MUT.
Genome_build: mm10
Supplementary_files_format_and_content: bed, motif and txt files of Plagl1

source_name: molar

growth_protocol: Mice were housed in pathogen-free conditions.

extract_protocol:
The apical thirds of their first molars were collected and single cells were isolated with collagenase, type 1.
Then, 5000 cells from each group were used for preparation of ATAC-seq libraries according to the protocol described by Kaestner Lab (https://www.med.upenn.edu/kaestnerlab/protocols.html).

treatment_protocol: Tamoxifen was dissolved in corn oil  at 20 mg/ml and injected intraperitoneally at a dosage of 1.5mg/10g body weight at PN3.5.

value: molar

tag: developmental stage

tag: strain

value: Arid1a fl/fl


title: cytotrophoblast cells-control-6h-rep3

description: Gene expression data from cytotrophoblast cells treated with vehicle for 6h rep3

source_name: Primary cytotrophoblast cells isolated from human term placenta treated with vehicle for 6h

value: villous tissue obtained from human term placenta

value: cultured cytotrophoblast cells

value: vehichle-control


title: cytotrophoblast cells-IL-11-6h-rep1

description: Gene expression data from cytotphoblast cells treated with 10ng/ml rhIL-11 for 6 h rep 1

value: female

value: term un-complicated pregnancy


title: WT1_S1


title: Control-1


title: ChIP HP1-CD Input repl2 in ovaries

status: Public on Apr 03 2021

city: Milano

first: Yinxiu

last: Zhan

address: Via Adamello 16

country: Italy

institute: European Institute for Oncology

data_processing:
Reads were mapped to the D. melanogaster genome (build dm6) using qAlign from QuasR package15 using the following alignments parameters: -n 2 -l 28 -e 70 -k 1 -X 500. This allows to keep reads mapping to multiple loci and randomly assigns them to one of the multiple locations. In case of Lambda DNA spike-in, the same parameters were used to map the unmapped reads to Lambda genome. ChIP enrichment over input was calculated using bamCompare from deepTools16  using the following parameters: --scaleFactorsMethod SES --smoothLength 900 --binSize 300. In case of spike-in normalization, the relative Dm6/lambda total number of reads for both input and IP was provided to bamCompare for normalisation through the option --scaleFactors. Peaks were called using macs217, using a cut-off of 0.05 on the q-value. --broad option was added for the H3K9me3 and HP1 ChIP.
Genome_build: dm6
Supplementary_files_format_and_content: bigwig file containing the enrichment of IP over input. In case of spikein, samples are first spike-in normalized and then the ratio between IP and input are taken

organism: Drosophila melanogaster

source_name: ChIP HP1-CD Input repl2 in ovaries

growth_protocol: Flies were reared under standard conditions and transferred to yeast two days prior ovary dissection.

extract_protocol:
Ovaries were snap frozen in batches of and stored at -80 ˚C until further processing.  Each batch was transferred to ice and homogenized for 1 min with an electric grinder in 50 µl of buffer A (60 mM KCl, 15 mM NaCl, 4 mM MgCl2, 15 mM HEPES [pH 7.6], 0.5 % TritonX100, 0.5 mM DTT, 10 mM Sodium Butyrate, 1X  Protease Inhibitors) supplemented with 1.8% PFA. The homogenate was filled up to 1 ml with buffer A + 1.8% PFA crosslinked for another 14 min rotating at room temperature. The crosslinking was stopped by the addition of 2.5 M glycine (250 mM final concentration). The nuclei were isolated by centrifugation and washed twice with buffer A. Subsequently the nuclei were resuspended in 100 µl Lysis buffer (140 mM NaCl, 15 mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 1 % TritonX100, 0.5 mM DTT, 0.1 % Sodium Deoxycholate, 10 mM Sodium Butyrate, 1% SDS, 0.5% N-Laurosylsarcosine,1X  Protease Inhibitors) and incubated for 2 hrs at 4 ˚C on a rocking plate. After that, the chromatin was sheared in a picoraptor (Diagenode) for a total of 16 cycles (30 sec on/30 sec off). Debris was pelleted at 20000 xg for 5 min at 4 ˚C and the supernatant was transferred to a new tube and diluted with 900 µl  Lysis buffer (w/o SDS) to a total SDS concentration of 0.1%.    Chromatin was precleared by addition of 50 μl of Protein A-Agarose (PA) suspension (Roche 11134515001) followed by overnight incubation at 4°C. PA was removed by centrifugation, antibodies (5 µl of rabbit anti-HA (C29F4) Cell Signaling) were added to the supernatant, and samples were incubated for 4 hr at 4°C in a rotating wheel. PA (50 μl) was added, and incubation was continued overnight at 4°C. Antibody-protein complexes were collected by centrifugation at 4,000 rpm for 1 min, and the supernatants were discarded. Samples were washed four times in Lysis buffer (with only 0.05% SDS) and twice in 1 mM EDTA, 10 mM Tris (pH 8) buffer (each wash, 5 min at 4°C). Chromatin was eluted from PA in 150 μl of 10 mM EDTA, 1% SDS, 50 mM Tris (pH 8) at 65°C for 15 min, followed by centrifugation and recovery of the supernatant. The eluate was incubated overnight at 65°C to reverse crosslinks and treated with Proteinase K for 3 hr at 50°C.  The chromatin was purified using ChIP DNA Clean & Concentrator columns (Zymo Research) and libraries were generated using the NEB Ultra II library preparation kit (E7645S).
libraries were prepared using NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturers instructions

treatment_protocol: The expression of the Flag-HA tagged transgenes (566 – WT HP1 and 575 – HP1 chromodomain mutant W45A Y48A) was induced by the driver line matalpha-tubulin (Bloomington #7063). In addition the expression of the shorthairpin RNA (line 571) against endogenous HP1 was controlled by the same driver (Bloomington #7063). In that way all cells expressing the HP1-transgene were depleted of endogenous HP1.

value: Ovaries

tag: chip antibody


title: ChIP HP1-CD IP repl2 in ovaries

source_name: ChIP HP1-CD IP repl2 in ovaries

value: rabbit anti-HA (C29F4) Cell Signaling)


title: normal tissue sample 1268

description:
PR1268
circularRNA expression in the tissue

value: normal tissue


title: normal tissue sample 1272

description:
PR1272
circularRNA expression in the tissue


title: ChIP HP1 Input repl1 in ovaries

source_name: ChIP HP1 Input repl1 in ovaries


title: ChIP HP1 Input repl2 in ovaries

source_name: ChIP HP1 Input repl2 in ovaries


title: cytotrophoblast cells-control-6h-rep2

description: Gene expression data from cytotrophoblast cells treated with vehicle for 6h rep2


title: cytotrophoblast cells-control-6h-rep1

description: Gene expression data from cytotrophoblast cells treated with vehicle for 6h rep1


title: MuT1_S2

value: Gli1-CreER;Arid1a fl/fl


title: ChIP HP1-CD Input repl1 in ovaries

source_name: ChIP HP1-CD Input repl1 in ovaries


description:
ChIP HP1 IP repl1 stg5 WT embryo with spike in
HP1_stg5_pooled_deepnorm_log2.bw

data_processing:
Reads were mapped to the D. melanogaster genome (build dm6) using qAlign from QuasR package15 using the following alignments parameters: -n 2 -l 28 -e 70 -k 1 -X 500. This allows to keep reads mapping to multiple loci and randomly assigns them to one of the multiple locations. In case of Lambda DNA spike-in, the same parameters were used to map the unmapped reads to Lambda genome. ChIP enrichment over input was calculated using bamCompare from deepTools16  using the following parameters: --scaleFactorsMethod SES --smoothLength 900 --binSize 300. In case of spike-in normalization, the relative Dm6/lambda total number of reads for both input and IP was provided to bamCompare for normalisation through the option --scaleFactors. Peaks were called using macs217, using a cut-off of 0.05 on the q-value. --broad option was added for the H3K9me3 and HP1 ChIP.
Genome_build: dm6
Supplementary_files_format_and_content: bigwig file containing the enrichment of IP over input. In case of spikein, samples are first spike-in normalised and then the ratio between IP and input are taken

growth_protocol: The flies were reared under standard conditions (25 C) and transferred to cages to collect the embryos for further experiments.

extract_protocol:
To extract the nuclei the embryos were transferred into Lysis buffer (140 mM NaCl, 15 mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 1 % TritonX100, 0.5 mM DTT, 0.1 % Sodium Deoxycholate, 10 mM Sodium Butyrate, 0.1% SDS, 0.5% N-Laurosylsarcosine,1X  Protease Inhibitors) and subjected to ultrasound treatment (Covaris E220, 45 sec, peak power 75, duty factor 10, cycles burst 200). The chromatin was sheared using Covaris E220 (900 sec, peak power 140, duty factor 5, cycle burst 200). The quality of the shearing was inspected on a Bioanalyzer 2100 (Agilent).  After sonication and 10 min high-speed centrifugation, fragmented chromatin was recovered in the supernatant. Chromatin was precleared by addition of 50 μl of Protein A-Agarose (PA) suspension (Roche 11134515001) followed by overnight incubation at 4°C. PA was removed by centrifugation, antibodies were added to the supernatant, and samples were incubated for 4 hr at 4°C in a rotating wheel. PA (50 μl) was added, and incubation was continued overnight at 4°C. Antibody-protein complexes were collected by centrifugation at 4,000 rpm for 1 min, and the supernatants were discarded. Samples were washed four times in Lysis buffer (with only 0.05% SDS) and twice in 1 mM EDTA, 10 mM Tris (pH 8) buffer (each wash, 5 min at 4°C). Chromatin was eluted from PA in 150 μl of 10 mM EDTA, 1% SDS, 50 mM Tris (pH 8) at 65°C for 15 min, followed by centrifugation and recovery of the supernatant. The eluate was incubated overnight at 65°C to reverse crosslinks and treated with Proteinase K for 3 hr at 50°C.  The chromatin was purified using ChIP DNA Clean & Concentrator columns (Zymo Research). In order to quantitatively assess the reduction of HP1 binding in the background of different mutants (HP1-KD and K9M) lambda DNA spike ins were added during the process. To perform a quantitative ChIP experiment all samples were processed in parallel starting from the same amount of embryos. After the preclearing a 20 µl aliquot of the sample was reverse crosslinked overnight at 65 °C and treated with RNAse and Proteinase K. The subsample was used to calculate the total amount of chromatin in the sample. All samples were then adjusted to contain the same amount of chromatin. Of the chromatin solution 10% were kept as an Input and lambda DNA was added at a final dilution of 1:1000 (usually around 60 pg of lambda DNA, when IPs were performed from 600 ng of total chromatin).  To account for differences in the amount of immunoprecipitated DNA 2 pg of lambda DNA per sample were added to the elution buffer. The lambda DNA was diluted in elution buffer and the same batch of buffer was used to elute all samples processed in parallel.
libraries were prepared using NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturers instructions

treatment_protocol: To collect precisely time staged embryos the flies were allowed to lay eggs on apple juice agar plates in cages for one hour at 25 °C. In order to synchronize the laying of the flies, plates were changed for three consecutive times and the embryos were discarded. After three pre-lays, the embryos were collected for one hours and aged for another two hours at 25 °C to reach the stage of zygotic genome activation. The plate with the embryos was removed from the incubator and the embryos were dechorionated with 50% bleach, washed with water and carefully dried. The embryos were transferred in 7 ml of heptane and 5 ml of freshly prepared fixative were added. The fixative consists of bufferof buffer A (60 mM KCl, 15 mM NaCl, 15 mM HEPES [pH 7.6], 4 mM MgCl2) and 1% of formaldehyde.  During the formaldehyde fixation the embryos were incubated on an orbital shaker at maximum speed for 15 min. The fixation was stopped by the addition of 225 mM glycine (final concentration). The embryos were incubated for 5 min on a wheel and washed with buffer A + 0.1% Triton. After washing, the embryos were hand sorted according to their typical morphology at each developmental stage on a cooling station under a microscope with transmitted light. The embryos were shock frozen and stored in batches of 400 at -80 °C.

value: WT embryo at stage 5

value: embryo (Bloomington #36303)

tag: antibody

value: HP1, Developmental Studies Hybridoma Bank C1A9, 2 µg


title: Control-2

tag: treatment


title: ChIP HP1 IP repl1 in ovaries

source_name: ChIP HP1 IP repl1 in ovaries


title: normal tissue sample 1255

description:
PR1255
circularRNA expression in the tissue

value: urothelial carcinoma of the bladder

value: male


description:
ChIP HP1 IP repl2 stg5 WT embryo with spike in
HP1_stg5_pooled_deepnorm_log2.bw


description:
ChIP HP1 Input repl2 stg5 WT embryo with spike in
HP1_stg5_pooled_deepnorm_log2.bw


description:
ChIP HP1 Input repl2 stg5 K9M embryo with spike in
HP1_in_K9M_pooled_deepnorm_log2.bw

value: K9M embryo at stage 5

value: embryo pUASP::H3.3K9M expression


title: ChIP HP1 IP repl2 in ovaries

source_name: ChIP HP1 IP repl2 in ovaries


description:
ChIP HP1 IP repl2 stg5 HP1KD embryo with spike in
HP1_in_HP1KD_pooled_deepnorm_log2.bw

value: HP1 KD embryo at stage 5

value: embryo HP1-KD (Bloomington #33400)


description:
ChIP HP1 Input repl1 stg5 K9M embryo with spike in
HP1_in_K9M_pooled_deepnorm_log2.bw


description:
ChIP HP1 Input repl2 stg5 HP1KD embryo with spike in
HP1_in_HP1KD_pooled_deepnorm_log2.bw


title: ChIP HP1-CD IP repl1 in ovaries

source_name: ChIP HP1-CD IP repl1 in ovaries


description:
ChIP HP1 Input repl1 stg5 WT embryo with spike in
HP1_stg5_pooled_deepnorm_log2.bw


description:
ChIP HP1 IP repl1 cycle 9-13 with spike in
HP1_c9_c13_pooled_deepnorm_log2.bw

value: WT embryo cycle 9-13


description:
ChIP HP1 IP repl1 stg5 HP1KD embryo with spike in
HP1_in_HP1KD_pooled_deepnorm_log2.bw


description:
ChIP HP1 IP repl2 cycle 9-13 with spike in
HP1_c9_c13_pooled_deepnorm_log2.bw


description:
ChIP HP1 Input repl1 stg5 HP1KD embryo with spike in
HP1_in_HP1KD_pooled_deepnorm_log2.bw


description:
ChIP HP1 Input repl2 cycle 9-13 with spike in
HP1_c9_c13_pooled_deepnorm_log2.bw


description:
ChIP HP1 IP repl2 stg5 K9M embryo with spike in
HP1_in_K9M_pooled_deepnorm_log2.bw


description:
ChIP HP1 Input repl1 before cycle 9 with spike in
HP1_before_c9_pooled_deepnorm_log2.bw

value: WT embryo before cycle 9


description:
ChIP HP1 IP repl2 before cycle 9 with spike in
HP1_before_c9_pooled_deepnorm_log2.bw


description:
ChIP HP1 Input repl2 before cycle 9 with spike in
HP1_before_c9_pooled_deepnorm_log2.bw


title: 2-RL-sham-gwat

city: Boston

first: Patrick

middle: J

last: Lenehan

address: 1 Jimmy Fund Way

institute: Dana Farber Cancer Institute

description: sham VAT 4

data_processing:
Transcript quantification was performed with Salmon, using quasi-mapping mode with selective alignment (validateMappings).
We set the rangeFactorizationBins to 4 and passed both the seqBias and gcBias flags to correct for sequence-specific bias and fragment-level GC bias, respectively.
Transcript quantifications were collapsed to gene-level quantifications using the tximport package and the Ensembl based annotation package EnsDb.Mmusculus.v79.
Differential expression analysis was performed using DESeq2 with fold change shrinkage by ashr.
For downstream analysis and visualization, only genes with a mean TPM (transcripts per million) > 2 in VAT from both sham-operated and tumor-bearing mice were considered.
Genome_build: mm10
Supplementary_files_format_and_content: comma separated file including transcripts per million (TPM) values from Salmon (sham_KPC_VAT_salmon_tpm_matrix.csv)
Supplementary_files_format_and_content: comma separated file including normalized counts from DESeq2 (sham_KPC_VAT_deseq_norm_count_matrix.csv)

source_name: Visceral adipose tissue

extract_protocol:
Adipose tissue was isolated from mice and immediately flash frozen in liquid nitrogen. Samples were then stored at -80oC. RNA was subsequently isolated using the QIAGEN RNeasy Plus Mini Kit (Qiagen #74134). Briefly, we added 350 ul of RLT Buffer supplemented with 3.5 ul β-mercaptoethanol (Sigma #M3148), and then crushed the tissue in a 1.5 mL tube using a plastic pestle. Samples were sequentially transferred to QIA Shredder columns, genomic DNA eliminator columns, and RNA isolation columns. RNA was eluted using 50 ul of nuclease-free water.
RNA libraries were prepared for sequencing at Genewiz using standard Illumina protocols

value: Visceral adipose tissue

value: 10 weeks

value: wild type

value: sham


description:
ChIP HP1 IP repl1 before cycle 9 with spike in
HP1_before_c9_pooled_deepnorm_log2.bw

value: WT embryo at stage before cycle 9


title: 1-NM-KPC-gwat

description: KPC VAT 1

value: orthotopic KPC pancreatic tumor


title: 4-NM-KPC-gwat

description: KPC VAT 4


title: 4-1R-sham-gwat

description: sham VAT 5


title: 1-1L-KPC-gwat

description: KPC VAT 2


title: 4-2R-KPC-gwat

description: KPC VAT 5


title: NET-seq_dTAG_rep2

first: Laurie

middle: A

last: Boyer

address: 77 Massachusetts Avenue

department: Biology

institute: Massachusetts Institute of Technology

data_processing:
library strategy: Native Elongating Transcript Sequencing (NET-Seq)
All the NET-seq FASTQ files were processed using custom Python scripts (https://github.com/BradnerLab/netseq). The sequencing reads are aligned to the mouse reference genome (mm10) using the STAR (v 2.7.2b) aligner (Dobin et al., 2013). PCR duplicates and reads arising from RT bias are also removed. Reads mapping exactly to the last nucleotide of each intron and exon (splicing intermediates) were further removed from the analysis. The final NET-seq BAM files were converted to bigwig, separated by strand, and normalized to SIRV spike-in controls using deepTools (v 3.0) with an “–offset 1” to record the position of the 5’ end of the sequencing read which corresponds to the 3’ end of the nascent RNA. NET-seq tags sharing the same or opposite orientation with the TSS were assigned as “sense” and “antisense” tags, respectively. Promoter-proximal regions were carefully selected for analysis to ensure that there is minimal contamination from transcription arising from other transcription units. Genes overlapping within a region of 2 kb upstream of the TSS were removed from the analysis.
Genome_build: mm10

growth_protocol: V6.5 murine embryonic stem (mES) cells were grown in 2i + LIF conditions. The media used for 2i + LIF media conditions is as follows: 484 mL DMEM/F12 (GIBCO 11320), 2.5 mL N2 supplement (GIBCO 17502048), 5 mL B27 supplement (GIBCO 17504044), 0.5 mM L-glutamine (GIBCO 25030), 1X non-essential amino acids (GIBCO 11140), 100 U/mL Penicillin-Streptomycin (GIBCO 15140), 0.1 mM β-mercaptoethanol (Sigma), 1 uM PD0325901 (Stemgent 04-0006), 3 uM CHIR99021 (Stemgent 04-0004), and 1000 U/mL recombinant LIF.

extract_protocol: The cell fractionation was performed as described previously (Mayer & Churchman, 2016). 10 million ES cells are washed with 500 ml of pre-cooled 1x PBS, resuspended in 150 ml Cytoplasmic lysis buffer (0.15% (v/v) NP-40, 10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25 mM a-amanitin (Sigma), 50 U SUPERase.In (Life Technologies), x1 Halt Protease Inhibitor cocktail mix (ThermoFisher 87786) and incubated on ice for 5 min. The cell lysate is layered over 400 ml of Sucrose buffer (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (w/v) sucrose, 25 mM a-amanitin, 50 U SUPERase.In, x1 Halt Protease Inhibitor cocktail mix) and centrifuged at 16,000 g for 10 min at 4C. The nuclei pellet is resuspended in 500 ml Nuclei wash buffer (0.1% (v/v) Triton X-100, 1 mM EDTA, in 1x PBS, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix) and centrifuged at 1,150 g for 1 min at 4C. Washed nuclei are resuspended in 200 ml Glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (v/v) glycerol, 0.85 mM DTT, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix). Next, 200 ml of Nuclei lysis buffer (1% (v/v) NP-40, 20 mM HEPES pH 7.5, 300 mM NaCl, 1M Urea, 0.2 mM EDTA, 1 mM DTT, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix) are added, mixed by pulsed vortexing and incubated on ice for 2 min. The lysate is centrifuged at 18,500 g for 2 min at 4C. The chromatin pellet is resuspended in 50 ml Chromatin resuspension solution (25 mM a-amanitin, 50 Units SUPERase.In, 1x Protease inhibitor mix in 1x PBS) before RNA preparation. Biological replicates were obtained from two independent Control and dTAG-13 treated cells. Prior to library preparation, SIRV spike-in controls (1:10,000 dilution) were added to the extracted RNA to account for loss of RNA during library preparation. RNA preparation and NET-seq library construction was conducted as originally described (Mylonas).  More than 90% recovery of ligated RNA and cDNA was achieved from 15% TBE-Urea (Invitrogen) and 10% TBE-Urea (Invitrogen), respectively, by adding RNA recovery buffer (R1070-1-10; Zymo Research) to the excised gel slices and further incubating at 70°C (1,500 rpm) for 15 min. Gel slurry was transferred through a ZymoSpin IV Column (C1007-50; Zymo Research) and further precipitated for subsequent library preparation steps. cDNA containing the 3’ end sequences of a subset of mature and heavily sequenced snRNAs, snoRNAs, and rRNAs were specifically depleted using biotinylated DNA oligos (Mylonas). Oligo-depleted circularized cDNA was amplified by PCR and double-stranded DNA was run on a 3% low melt agarose gel. The final NET-seq library running at ~150 bp was extracted and further purified using the ZymoClean Gel DNA recovery kit (Zymo Research). Sample purity and concentration was assessed in a 2200 TapeStation and further deep sequenced in a HiSeq 2500 Illumina Platform  The cell fractionation was performed as described previously (Mayer & Churchman, 2016). 10 million ES cells are washed with 500 ml of pre-cooled 1x PBS, resuspended in 150 ml Cytoplasmic lysis buffer (0.15% (v/v) NP-40, 10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25 mM a-amanitin (Sigma), 50 U SUPERase.In (Life Technologies), x1 Halt Protease Inhibitor cocktail mix (ThermoFisher 87786) and incubated on ice for 5 min. The cell lysate is layered over 400 ml of Sucrose buffer (10 mM Tris-HCl (pH 7.0), 150 mM NaCl, 25% (w/v) sucrose, 25 mM a-amanitin, 50 U SUPERase.In, x1 Halt Protease Inhibitor cocktail mix) and centrifuged at 16,000 g for 10 min at 4C. The nuclei pellet is resuspended in 500 ml Nuclei wash buffer (0.1% (v/v) Triton X-100, 1 mM EDTA, in 1x PBS, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix) and centrifuged at 1,150 g for 1 min at 4C. Washed nuclei are resuspended in 200 ml Glycerol buffer (20 mM Tris-HCl (pH 8.0), 75 mM NaCl, 0.5 mM EDTA, 50% (v/v) glycerol, 0.85 mM DTT, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix). Next, 200 ml of Nuclei lysis buffer (1% (v/v) NP-40, 20 mM HEPES pH 7.5, 300 mM NaCl, 1M Urea, 0.2 mM EDTA, 1 mM DTT, 25 mM a-amanitin, 50 U SUPERase.In, 1x Protease inhibitor mix) are added, mixed by pulsed vortexing and incubated on ice for 2 min. The lysate is centrifuged at 18,500 g for 2 min at 4C. The chromatin pellet is resuspended in 50 ml Chromatin resuspension solution (25 mM a-amanitin, 50 Units SUPERase.In, 1x Protease inhibitor mix in 1x PBS) before RNA preparation. Biological replicates were obtained from two independent Control and dTAG-13 treated cells. Prior to library preparation, SIRV spike-in controls (1:10,000 dilution) were added to the extracted RNA to account for loss of RNA during library preparation. RNA preparation and NET-seq library construction was conducted as originally described (Mylonas).  More than 90% recovery of ligated RNA and cDNA was achieved from 15% TBE-Urea (Invitrogen) and 10% TBE-Urea (Invitrogen), respectively, by adding RNA recovery buffer (R1070-1-10; Zymo Research) to the excised gel slices and further incubating at 70°C (1,500 rpm) for 15 min. Gel slurry was transferred through a ZymoSpin IV Column (C1007-50; Zymo Research) and further precipitated for subsequent library preparation steps. cDNA containing the 3’ end sequences of a subset of mature and heavily sequenced snRNAs, snoRNAs, and rRNAs were specifically depleted using biotinylated DNA oligos (Mylonas). Oligo-depleted circularized cDNA was amplified by PCR and double-stranded DNA was run on a 3% low melt agarose gel. The final NET-seq library running at ~150 bp was extracted and further purified using the ZymoClean Gel DNA recovery kit (Zymo Research). Sample purity and concentration was assessed in a 2200 TapeStation and further deep sequenced in a HiSeq 2500 Illumina Platform

tag: cell type

value: Embryonic stem cells

tag: genotype/variation

tag: molecule subtype

value: nascent RNA


title: 1-RL-sham-gwat

description: sham VAT 2

tag: age


title: 2-1R-sham-gwat

description: sham VAT 3

tag: genotype


title: 1-1R-sham-gwat

description: sham VAT 1


title: NET-seq_dTAG_rep1


title: NET-seq_Control_rep2


title: ChIP-seq_Input_rep2

data_processing:
Reads were aligned to the mm10 genome using Bowtie2 (v 2.3.5) (Langmead & Salzberg, 2012). Peak calling was performed using PePr (v 1.1) (Zhang et al, 2014) with peaks displaying an FDR < 10−5 considered statistically significant. All aligned ChIP-seq BAM files were converted to bigwig (10 bp bin) and normalized to 1× sequencing depth using deepTools (v 3.0) (Ramirez et al, 2016). Blacklisted mm10 coordinates were further removed from the analysis. Average binding profiles were visualized using R (v 3.6.1). Heatmaps were generated with deepTools.
Genome_build: mm10

extract_protocol: ChIP was performed in ~10 million ES cells, per assay, as previously described (Mylonas). Briefly, cells were cross-linked with 1% formaldehyde for 10 min followed by by 5 min quenching with 125 mM glycine. After washing with PBS buffer, the cells were collected and lysed in cell lysis buffer (5 mM Tris, pH 8.0, 85 mM KCl, and 0.5% NP-40) with x1 Halt Protease Inhibitor cocktail (ThermoFisher 87786) and 1mM PMSF (Sigma 10837091001). Pellets were spun for 5 min at 6000 rpm at 4°C. Nuclei were lysed in nuclei lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris–HCl) and samples were sonicated for 12 min on a Covaris Sonicator. The samples were centrifuged for 20 min at 13,000 rpm at 4°C and the supernatant was diluted in IP buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, and 167 mM NaCl), and the appropriate antibody (10 μg) was added and incubated overnight at 4°C with rotation. Antibodies used in this study; Anti-HA (Cell Signalling 3724), Anti-Spt5 (Santa Cruz, sc-133217), Anti-Dendra2 (OriGene TA180094). Two biological replicates were prepared for each condition using independent cell cultures and chromatin precipitations. The next day, 50 μl Protein G Dynabeads (Life Technologies #10009D) were added for 1 h at room temperature with rotation. Beads were washed once for 1 min with rotation with each of the following buffers: Low salt buffer (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris pH 8.0, 150mM NaCl), High salt buffer (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl), LiCl buffer (0.25M LiCL, 1% NP-40, 1% dioxycholate, 10mM Tris pH 8.0, 1mM EDTA), and TE buffer (50mM Tris pH 8.0 , 10mM  EDTA). DNA was eluted off the beads by rotation at room temperature for 30 min in 200 μL elution buffer (1% SDS, 0.1M NaHCO3). Cross-links were reversed at 65°C for 4h. RNA was degraded by the addition of 3 μL of 33 mg/mL RNase A (Sigma, R4642) and incubation at 37°C for 2 hours. Protein was degraded by the addition of 5 μL of 20 mg/mL proteinase K (Invitrogen, 25530049) and incubation at 50°C for 1 hour. A phenol:chloroform:isoamyl alcohol extraction was performed followed by an ethanol precipitation. The DNA was then resuspended in 20 μL H2O and used for either qPCR or sequencing.  For ChIP-seq experiments, purified 10-20 ng of ChIP DNA was used to prepare Illumina multiplexed sequencing libraries. Libraries for Illumina sequencing were prepared following the NEBNext DNA Library Prep Master Mix kit (NEB E6040). Amplified libraries were size-selected using a 2% agarose gel to extract fragments between 200 and 600 bp.


title: NET-seq_Control_rep1


description:
ChIP HP1 Input repl1 cycle 9-13 with spike in
HP1_c9_c13_pooled_deepnorm_log2.bw

value: embryo (Bloomington #36303)


title: ChIP-seq_H2A.Z.1-HA_Control_rep1

value: Anti-HA (Cell Signalling 3724)


title: ChIP-seq_H2A.Z.1-HA_Control_rep2


title: ChIP-seq_RNAPII_Anti-Dendra2

value: H2afz-FKBP-V + Dendra2-RNAPII

value: Anti-Dendra2 (OriGene TA180094)


title: ChIP-seq_H2A.Z.1-HA_dTAG_rep2


title: ChIP-seq_Input_rep1


title: 2-2R-KPC-gwat

description: KPC VAT 3


title: ChIP-seq_H2A.Z.1-HA_dTAG_rep1


title: 22mi

city: Houston

first: Sean

last: Hartig

state: TX

address: One Baylor Plaza, BCM130

department: Molecular and Cellular Biology

institute: Baylor College of Medicine

data_processing:
Reads were mapped with TopHat 2.0. Only uniquely mapping reads were allowed.
The mapped reads were then assembled into transcripts with Cufflinks 2.0 and the UCSC RefSeq gene model.
Gene expression (FPKM) was quantile normalized using the R statistical system.
Genome_build: UCSC mm10/NCBI 38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample

source_name: inguinal WAT infected with miR-30a adenovirus that also expresses a GFP reporter gene

extract_protocol:
Total RNA was extracted using Direct-Zol (Zymo).  Libraries were sequenced and the library was amplified for sequencing.  Sequencing was performed on the Illumina HiSeq2000.
RNA libraries were prepared for sequencing using standard Illumina protocols

tag: diet

value: 60% high fat diet for >12 weeks


title: 20mi


title: 21mi


title: 18mi


title: 16C

source_name: inguinal WAT infected with GFP control adenovirus


title: Donor 0967, NGFR-transduced Tconv + none stimulation

city: Vancouver

first: Megan

middle: K

last: Levings

address: 950 West 28th Ave

country: Canada

institute: University of British Columbia

description:
Batch: 2
48R221_S7

data_processing:
Within Illumina BaseSpace RNA-Seq Alignment App (v1.1.0): base-calls made with RTA (v2.1), demultiplexing with bcl2fastq2, read sequences aligned to hg19 reference genome with STAR (v2.5.0a) and lentiviral transgene sequences using Burrows-Wheeler Aligner (v.0.7.10).
Reads that mapped to lentiviral transgene sequences were removed from further analysis.
From raw counts, library scale factors were calculated from edgeR (v3.24.3), then normalized using limma (v3.38.3) within R (accounting for donor/sex variation) to identify differentially expressed genes.
In R, cross-referenced differentially expressed genes with .GMT databases from GSEA MSigDB Collections (hallmark gene sets, GO gene sets, transcription factor targets)
Genome_build: hg19
Supplementary_files_format_and_content: Per sample: raw counts of all genes, generated by HTSeq (v0.11.2)

molecule: polyA RNA

source_name: Peripheral blood bulk Treg

growth_protocol: Day 0: Bulk Tregs (CD4+CD25hiCD127lo) and bulk Tconv (CD4+CD25loCD127hi) were flow-sorted and expanded 1:1 with irradiated L cells + OKT3 (0.1μg/ml) + IL-2 (1000IU/mL). Day 1: Cell were transduced with a lentiviral vector encoding NGFR and the denoted CAR construct (MOI = 10). Day 7: NGFR+ cells were bead-purified by positive selection and rested overnight in IL-2 (100IU/mL). Cultured in Immunocult-XF T cell media, 1% (v/v) penicillin-streptomycin.

extract_protocol:
Activated cells were pelleted (500g for 5 min), washed once with 1x PBS, then processed immediately for RNA using NEB RNA Miniprep Kit (T2010S). All samples used in this study had an RNA integrity number > 8.
mRNA enrichment and library preparation with NEBNext Poly(A) mRNA Magnetic Isolation Module, NEBNExt Ultra II Directional RNA Library Prep Kit for Illumina.

treatment_protocol: Day 8: 250 000 rested, expanded Tregs/Tconv were stimulated with either no beads, 125 000 HLA-A*02:01-coated beads OR 250 000 anti-CD3/28 Dynabeads in Immunocult-XF T cell media, 1% (v/v) penicillin-streptomycin for 24 hours.

tag: donor

tag: Sex

value: F

value: Tconv

tag: transduction

value: NGFR

tag: expansion time

value: 7 days

tag: culture condition

tag: culture time


title: 14C


title: Donor 0967, NGFR-transduced Treg + none stimulation

description:
Batch: 2
47R221_S6

value: Treg


title: 13C


title: Donor 0967, CD28wt-NGFR-transduced Tconv + CAR stimulation

description:
Batch: 2
46R221_S19

value: CAR


title: Donor 0967, NGFR-transduced Tconv + TCR stimulation

description:
Batch: 2
45R221_S18

value: TCR


title: Donor 0967, CD3zeta-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
43R221_S16


title: Donor 0967, TNFR-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
41R221_S14

value: TNFR-NGFR


title: Donor 0967, 41BB-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
40R221_S13


title: Donor 0967, CTLA4wt-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
44R221_S17


title: Donor 0967, GITR-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
39R221_S12

value: GITR-NGFR


title: Donor 0967, CTLA4mut-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
42R221_S15


title: Donor 0967, CD28mut-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
38R221_S23


title: Donor 0967, OX40-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
35R221_S4


title: 15C


title: Donor 0967, CD28wt-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
34R221_S21


title: Donor 0967, PD1-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
37R221_S5


title: Donor 0967, NGFR-transduced Treg + TCR stimulation

description:
Batch: 2
33R221_S20


title: Donor 0960, NGFR-transduced Tconv + none stimulation

description:
Batch: 2
32R221_S3

value: M


title: Donor 0960, NGFR-transduced Treg + none stimulation

description:
Batch: 2
31R221_S2


title: Donor 0960, CTLA4wt-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
28R221_S8


title: Donor 0960, CD28wt-NGFR-transduced Tconv + CAR stimulation

description:
Batch: 2
30R221_S19


title: Donor 0960, NGFR-transduced Tconv + TCR stimulation

description:
Batch: 2
29R221_S18


title: Donor 0960, CTLA4mut-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
26R221_S7


title: Donor 0960, CD3zeta-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
27R221_S16


title: Donor 0960, 41BB-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
24R221_S5


title: Donor 0960, TNFR-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
25R221_S14


title: Donor 0960, GITR-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
23R221_S4


title: Donor 0960, CD28mut-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
22R221_S11


title: Donor 0960, PD1-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
21R221_S1


title: Donor 0960, CD28wt-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
18R221_S8


title: Donor 0960, ICOS-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
20R221_S10

value: ICOS-NGFR


title: Donor 0960, OX40-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
19R221_S9


title: Donor 0960, NGFR-transduced Treg + TCR stimulation

description:
Batch: 2
17R221_S20


title: Donor 9649, NGFR-transduced Tconv + TCR stimulation

description:
Batch: 1
13R221_S22


title: Donor 9649, CD28wt-NGFR-transduced Tconv + CAR stimulation

description:
Batch: 1
14R221_S23


title: Donor 0967, ICOS-NGFR-transduced Treg + CAR stimulation

description:
Batch: 2
36R221_S11


title: Donor 9649, CD3zeta-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
11R221_S20


title: Donor 9649, NGFR-transduced Treg + none stimulation

description:
Batch: 1
15R221_S24


title: Donor 9649, NGFR-transduced Tconv + none stimulation

description:
Batch: 1
16R221_S1


title: Donor 9649, CTLA4mut-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
10R221_S19


title: Donor 9649, TNFR-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
9R221_S18


title: Donor 9649, GITR-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
7R221_S16


title: Donor 9649, 41BB-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
8R221_S17


title: Donor 9649, PD1-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
5R221_S14


title: Donor 9649, OX40-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
3R221_S12


title: Donor 9649, CD28mut-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
6R221_S15


title: Donor 9649, NGFR-transduced Treg + TCR stimulation

description:
Batch: 1
1R221_S10


title: Donor 9649, CD28wt-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
2R221_S11


title: ATAC-seq_Control_rep2

data_processing:
Sequenced paired mates were mapped either to mm10 or hg19 genome build using Bowtie2 (v 2.3.5) with the following parameters: –X 2000. Mapped reads were shifted and split to either “Nucleosome free” or “Mono-nucleosome” reads using the “ATACseqQC”9 R package. All ATAC-seq BAM files were converted to bigwig, binned (10 bp), and normalized to spike-in controls using deepTools (v 3.0). Blacklisted mm10 coordinates were further removed from the analysis. Average binding profiles were visualized using R (v 3.6.1). For computing normalization factors from spike-in controls, we calculated the number of all reads that uniquely mapped to the human genome, at 10kbp bin intervals, using “multiBamSummary”. Counts were parsed into R and DESeq was used to calculate normalization factors. The inverse of the normalization factor was applied on all mouse mapped reads upon bigwig conversion using the “--scaleFactor” option implemented in the “bamCoverage” command of deeptools (v 3.0).
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

source_name: Embryonic stem cells

extract_protocol: ATAC-seq was performed on 80,000 mESCs, along with 5% spike-in U2OS human cells, using the ATAC-seq kit (Active Motif) according to the manufacturer’s instructions.

value: Control


title: ATAC-seq_dTAG_rep2


title: ATAC-seq_dTAG_rep1


title: ChIP-nexus_Spt5_dTAG_rep2

data_processing:
Library strategy: ChIP-nexus
Fastq files were filtered for the presence of the four mixed barcodes and were deduplicated using Q-Nexus (Hansen et al., 2016). Bowtie2 (v 2.3.5) was used for alignment to either the mm10 or hg19 genome. Normalization factors were computed for reads mapping uniquely to the human genome using Deseq2 (Love, Huber, & Anders, 2014). All aligned mouse ChIP-nexus BAM files were converted to bigwig (1 bp bin), separated by strand, and normalized to human spike-in controls using deepTools (v 3.0) with an “–offset 1” to record the position of the 5’ end of the sequencing read which corresponds to TF footprint.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

extract_protocol: DNA-protein-complexes were isolated with specific antibodies when still being attached to protein G-dynabeads. DNA was end repaired using NEBNext End repair module (#E6050L). A single 3'-A overhang was added to the blunted ends using NEBNext dA-Tailing module (#E6053L). Adapters with single 3'-T overhangs and 5’ overhangs (barcode composed of 5 random nucleotides) were ligated to the adenylated fragments. Ligated fragments were extended with Phi29 DNA polymerase and digested by lambda exonuclease. Digested single strand DNA was then eluted, reverse cross-linked and purified prior to self-circularization by Circligase. Linearized single strand DNA was then PCR-amplified using adaptor sequences and library was purified on 2% agarose gel to remove adaptor-adaptor ligation products. Following cluster generation on the flowcell surface, the sequencing libraries were sequenced following Illumina's protocol.

value: Anti-Spt5 (Santa Cruz sc-133217)


title: ATAC-seq_Control_rep1


title: ChIP-nexus_Spt5_dTAG_rep1


title: ChIP-nexus_Spt5_Control_rep1


title: ChIP-nexus_Spt5_Control_rep2


title: T1D5_HPAP032_scRNA-seq

city: Philadelphia

first: Maria

last: Fasolino

address: 324 BRB II/III, 421 Curie Blvd

institute: University of Pennsylvania

data_processing:
Cell Ranger (10x Genomics; 2.1.0) was used for bcl2fastq conversion.
Cell Ranger (10x Genomics; 2.1.0) was used for aligning, filtering, counting, and cell calling.
Samples were aggregated using Cell Ranger (10x Genomics; 2.1.0).
Seurat (3.0.2; (Butler et al., 2018; Stuart et al., 2019)) was used for filtering, UMAP generation, and initial clustering.
DoubletFinder (McGinnis et al., 2019) was used to demarcate and remove potential doublets.
Genome_build: TooManyCells was then used to cluster and visualize the 34,259 single cells.

source_name: Bulk human pancreatic islet cells

extract_protocol:
Organs were recovered and processed as previously described (Campbell-Thompson et al., 2012).
The Single Cell 3’ Reagent Kit v2 or v3 (10x) was used for generating scRNA-seq data.

tag: tissue

value: human pancreatic islet cells

tag: disease state

value: type 1 diabetes


title: ChIP-nexus_NELF-B-GFP_TRI_rep2

tag: strain

tag: chip antibody

value: Anti-GFP (Novus Biologicals NB600-308)

value: H2afz-FKBP-V + Nelf-b-GFP

value: Triptolide


title: ChIP-nexus_NELF-B-GFP_TRI_rep1

tag: treatment


title: T1D3_HPAP023_scRNA-seq


title: T1D2_HPAP021_scRNA-seq


title: Donor 9649, CTLA4wt-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
12R221_S21

value: F

tag: cell type


title: ChIP-nexus_NELF-B-GFP_DRB_rep2

value: DRB


title: AAB3_HPAP029_scRNA-seq

value: AAB+


title: T1D1_HPAP020_scRNA-seq


title: ChIP-nexus_NELF-B-GFP_DRB_rep1


title: ChIP-nexus_NELF-B-GFP_dTAG_rep1


title: Donor 9649, ICOS-NGFR-transduced Treg + CAR stimulation

description:
Batch: 1
4R221_S13

value: Treg

value: 7 days


title: AAB1_HPAP019_scRNA-seq


title: ChIP-nexus_NELF-B-GFP_Control_rep2


title: Control6_HPAP026_scRNA-seq


title: ChIP-seq_H3_dTAG_rep2

data_processing:
Reads were aligned to the mm10 genome using Bowtie2 (v 2.3.5) (Langmead & Salzberg, 2012). Peak calling was performed using PePr (v 1.1) (Zhang et al, 2014) with peaks displaying an FDR < 10−5 considered statistically significant. All aligned ChIP-seq BAM files were converted to bigwig (10 bp bin) and normalized to 1× sequencing depth using deepTools (v 3.0) (Ramirez et al, 2016). Blacklisted mm10 coordinates were further removed from the analysis. Average binding profiles were visualized using R (v 3.6.1). Heatmaps were generated with deepTools.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig

extract_protocol: ChIP was performed in ~10 million ES cells, per assay, as previously described (Mylonas & Tessarz, 2018). Briefly, cells were cross-linked with 1% formaldehyde for 10 min followed by by 5 min quenching with 125 mM glycine. After washing with PBS buffer, the cells were collected and lysed in cell lysis buffer (5 mM Tris, pH 8.0, 85 mM KCl, and 0.5% NP-40) with x1 Halt Protease Inhibitor cocktail (ThermoFisher 87786) and 1mM PMSF (Sigma 10837091001). Pellets were spun for 5 min at 6000 rpm at 4°C. Nuclei were lysed in nuclei lysis buffer (1% SDS, 10 mM EDTA, and 50 mM Tris–HCl) and samples were sonicated for 12 min on a Covaris Sonicator. The samples were centrifuged for 20 min at 13,000 rpm at 4°C and the supernatant was diluted in IP buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl, and 167 mM NaCl), and the appropriate antibody (10 μg) was added and incubated overnight at 4°C with rotation. Antibodies used in this study; Anti-HA (Cell Signalling 3724), Anti-Spt5 (Santa Cruz, sc-133217), Anti-Dendra2 (OriGene TA180094). Two biological replicates were prepared for each condition using independent cell cultures and chromatin precipitations. The next day, 50 μl Protein G Dynabeads (Life Technologies #10009D) were added for 1 h at room temperature with rotation. Beads were washed once for 1 min with rotation with each of the following buffers: Low salt buffer (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris pH 8.0, 150mM NaCl), High salt buffer (0.1% SDS, 1% Triton-X-100, 2mM EDTA, 20mM Tris pH 8.0, 500mM NaCl), LiCl buffer (0.25M LiCL, 1% NP-40, 1% dioxycholate, 10mM Tris pH 8.0, 1mM EDTA), and TE buffer (50mM Tris pH 8.0 , 10mM EDTA). DNA was eluted off the beads by rotation at room temperature for 30 min in 200 μL elution buffer (1% SDS, 0.1M NaHCO3). Cross-links were reversed at 65°C for 4h. RNA was degraded by the addition of 3 μL of 33 mg/mL RNase A (Sigma, R4642) and incubation at 37°C for 2 hours. Protein was degraded by the addition of 5 μL of 20 mg/mL proteinase K (Invitrogen, 25530049) and incubation at 50°C for 1 hour. A phenol:chloroform:isoamyl alcohol extraction was performed followed by an ethanol precipitation. The DNA was then resuspended in 20 μL H2O and used for either qPCR or sequencing. For ChIP-seq experiments, purified 10-20 ng of ChIP DNA was used to prepare Illumina multiplexed sequencing libraries. Libraries for Illumina sequencing were prepared following the NEBNext DNA Library Prep Master Mix kit (NEB E6040). Amplified libraries were size-selected using a 2% agarose gel to extract fragments between 200 and 600 bp.

value: Anti-H3 (Abcam ab1791)


title: ChIP-nexus_NELF-B-GFP_Control_rep1


title: Control5_HPAP037_scRNA-seq


title: Control3_HPAP035_scRNA-seq


title: ChIP-seq_H3_Control_rep2


title: ChIP-seq_H3_dTAG_rep1


title: T1D4_HPAP028_scRNA-seq


title: ChIP-seq_H3_Control_rep1


title: Control4_HPAP036_scRNA-seq


title: ChIP-seq_H2A.Z.2_FLAG_dTAG_rep2

value: Anti-FLAG (Cell Signalling 14793)

value: H2afz-FKBP-V + H2afv-3xFLAG


title: Control1_HPAP022_scRNA-seq


title: ChIP-seq_H2A.Z.2_FLAG_dTAG_rep1


title: ChIP-seq_H2A.Z.2_FLAG_Control_rep2


title: ChIP-nexus_NELF-B-GFP_dTAG_rep2


title: ChIP-seq_H2A.Z.2_FLAG_Control_rep1


title: AAB2_HPAP024_scRNA-seq


title: 70-SRSF9 KD_UN_replicate 1

city: Bryan

first: Kristin

middle: L

last: Patrick

state: Texas

address: 8447 Riverside Pkwy, MREB 3102

department: Microbial Pathogenesis and Immunology

institute: Texas A&M University, College of Medicine

data_processing:
2x150 paired-end sequence reads
Illumina software used for basecalling
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to Ensemble Grcm38 with CLC genomics Workbench
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using CLC Genomics Workbench
Genome_build: Grcm38
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each Sample

source_name: RAW 264.7 Macrophage

extract_protocol:
RNA was harvested using Trizol reagent. Zymo Research Direct-zol kits were used to isolate RNA with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

treatment_protocol: RAW 264.7 Macrophages seeded in a 12 well dish at 8x10^5 cells/well overnight. The next day cells were ~80% confluent. For baseline (UN) measurements, cells were collected without treatment. For infected (SAL) samples, cells were infected with Salmonella Tyhpimurium (SAL) (MOI = 10) and collected at 4h post-infection.

value: SRSF9 KD shRNA Knockdown

tag: cell line

value: no treatment


title: 71-SRSF9 KD_UN_replicate 2


title: ChIP-seq_RNAPII_washoff_rep1

tag: antibody

value: Anti-Dendra2 (OriGene TA180094)

value: H2afz-FKBP-V + Dendra2-RNAPII

value: DRB wash-off


title: 68-SRSF9 KD_SAL_replicate 2

value: Salmonella infection


title: ChIP-seq_RNAPII_washoff_rep2


title: 67-SRSF9 KD_SAL_replicate 1


title: 72-SRSF9 KD_UN_replicate 3


title: ChIP-seq_RNAPII_DRB_rep2


title: 69-SRSF9 KD_SAL_replicate 3


title: ChIP-seq_RNAPII_DRB_rep1


title: 65-SRSF7 KD_UN_replicate 2

value: SRSF7 KD shRNA Knockdown


title: ChIP-seq_RNAPII_DMSO_rep2

value: DMSO


title: 66-SRSF7 KD_UN_replicate 3


title: ChIP-seq_RNAPII_DMSO_rep1


title: 64-SRSF7 KD_UN_replicate 1


title: Control2_HPAP034_scRNA-seq

value: human pancreatic islet cells

value: Control


title: ChIP-seq_H2A.Z_washoff_rep2

value: Anti-HA (Cell Signalling 3724)


title: 63-SRSF7 KD_SAL_replicate 3


title: ChIP-seq_H2A.Z_washoff_rep1


title: ChIP-seq_H2A.Z_DRB_rep2

value: DRB


title: 62-SRSF7 KD_SAL_replicate 2


title: 61-SRSF7 KD_SAL_replicate 1


title: 60-SRSF6 KD_UN_replicate 3

value: SRSF6 KD shRNA Knockdown


title: ChIP-seq_H2A.Z_DRB_rep1


title: ChIP-seq_H2A.Z_DMSO_rep1


title: 59-SRSF6 KD_UN_replicate 2


title: 58-SRSF6 KD_UN_replicate 1


title: ChIP-nexus_TFIIB_dTAG_TRI40min_rep2

data_processing:
Fastq files were filtered for the presence of the four mixed barcodes and were deduplicated using Q-Nexus (Hansen et al., 2016).  Bowtie2 (v 2.3.5) was used for alignment to either the mm10 or hg19 genome. Normalization factors were computed for reads mapping uniquely to the human genome using Deseq2 (Love, Huber, & Anders, 2014). All aligned mouse ChIP-nexus BAM files were converted to bigwig (1 bp bin), separated by strand, and normalized to human spike-in controls using deepTools (v 3.0) with an “–offset 1” to record the position of the 5’ end of the sequencing read which corresponds to TF footprint.
Genome_build: mm10
Supplementary_files_format_and_content: Bigwig

extract_protocol: DNA-protein-complexes were isolated with specific antibodies when still being attached to protein G-dynabeads. DNA was end repaired using NEBNext End repair module (#E6050L). A single 3'-A overhang was added to the blunted ends using NEBNext dA-Tailing module (#E6053L). Adapters with single 3'-T overhangs and 5’ overhangs (barcode composed of 5 random nucleotides) were ligated to the adenylated fragments. Ligated fragments were extended with Phi29 DNA polymerase and digested by lambda exonuclease. Digested single strand DNA was then eluted, reverse cross-linked and purified prior to self-circularization by Circligase.  Linearized single strand DNA was then PCR-amplified using adaptor sequences and library was purified on 2% agarose gel to remove adaptor-adaptor ligation products. Following cluster generation on the flowcell surface, the sequencing libraries were sequenced following Illumina's protocol.

value: Embryonic stem cells

tag: genotype/variation

value: dTAG + Triptolide 40 min

value: Anti-TFIIB (Cell Signaling, 4169)


title: ChIP-nexus_TFIIB_dTAG_TRI40min_rep1


title: 57-SRSF6 KD_SAL_replicate 3

tag: genotype


title: 54-SRSF2 KD_UN_replicate 3

value: SRSF2 KD shRNA Knockdown


title: ChIP-nexus_TFIIB_dTAG_rep1

value: dTAG + no drug


title: 52-SRSF2 KD_UN_replicate 1

value: no treatment


title: ChIP-nexus_TFIIB_Control_TRI40min_rep1

value: Control + Triptolide 40 min


title: ChIP-nexus_TFIIB_Control_TRI40min_rep2


title: 55-SRSF6 KD_SAL_replicate 1


title: ChIP-nexus_TFIIB_Control_rep1

value: Control + no drug


title: 53-SRSF2 KD_UN_replicate 2


title: 51-SRSF2 KD_SAL_replicate 3


title: ChIP-nexus_TFIIB_Control_rep2


title: 50-SRSF2 KD_SAL_replicate 2


title: ChIP-nexus_TBP_dTAG_TRI40min_rep2

value: Anti-TBP (Abcam, ab28175)


title: ChIP-nexus_TBP_dTAG_TRI40min_rep1


title: 48-SRSF1 KD_UN_replicate 3

value: SRSF1 KD shRNA Knockdown


title: ChIP-seq_H2A.Z_DMSO_rep2

value: H2afz-FKBP-V + Dendra2-RNAPII


title: ChIP-nexus_TBP_dTAG_rep2


title: ChIP HP1 IP repl2 Covance

description:
ChIP HP1 IP repl2 Covance
HP1_covance_deepnorm_log2_smooth.bw

source_name: ChIP HP1 IP repl2 Covance

value: WT embryo at stage 5

value: embryo (Bloomington #36303)

value: HP1, Covance, PRB-291C-200, 1 µg


title: 47-SRSF1 KD_UN_replicate 2


title: ChIP HP1 IP repl1 Covance

description:
ChIP HP1 IP repl1 Covance
HP1_covance_deepnorm_log2_smooth.bw

source_name: ChIP HP1 IP repl1 Covance


title: ChIP-nexus_TBP_dTAG_rep1


title: ChIP HP1 Input repl1 Covance

description:
ChIP HP1 Input repl1 Covance
HP1_covance_deepnorm_log2_smooth.bw

source_name: ChIP HP1 Input repl1 Covance


description:
ChIP HP1 IP repl1 stg5 K9M embryo with spike in
HP1_in_K9M_pooled_deepnorm_log2.bw

value: K9M embryo at stage 5

value: embryo pUASP::H3.3K9M expression

value: HP1, Developmental Studies Hybridoma Bank C1A9, 2 µg


title: ChIP-nexus_TFIIB_dTAG_rep2

value: Embryonic stem cells


title: 56-SRSF6 KD_SAL_replicate 2

value: SRSF6 KD shRNA Knockdown

value: Salmonella infection


title: HiC HP1 KD rnai2 S2 cells repl 2

description: HiC HP1 KD rnai2 S2 cells repl 2

data_processing:
Hi-C paired end datasets were processed using HiC-Pro (version 2.11.1). HiC-Pro covers the whole analysis from fastq files to the interaction matrix
Genome_build: dm6
Supplementary_files_format_and_content: iced and binned matrix (10kb). The format of the matrix is the standard from HiC-Pro, namely, 3 columns: start end interactions. It's a sparce matrix annotation, no zero values are reported.

source_name: HiC HP1 KD rnai2 S2 cells repl 2

growth_protocol: Cells were seeded in Express Five SFM (Gibco 10486025) supplemented with GlutaMAX (Gibco 35050061) at 1 Mio cells/ml in 6 ml shaking flasks and grown for 4 days to reach a total of approximately 40 Mio cells (6-7 Mio cells/ml).

extract_protocol:
For S2 cells the nuclei were released using the NEXSON protocol. Briefly, the cells were resuspended in 1 ml of ice-cold HiC lysis buffer and subjected to ultrasound in a Covaris sonicator (using the following parameters peak power 75, duty factor 2, cycles/burst 200, 67 sec). The integrity of the nuclei was checked under the microscope.  The nuclei were pelleted for 5 min at 3000 g (rcf) and 4 °C. After discarding the supernatant, the pellet was incubated in 50 µl 0.5% SDS for 10 min at RT. The reaction was quenched by adding 145 µl of water and 25 µl of 10% Triton X100.   To digest the chromatin 25 µl of DpnII buffer were added and 0.7 µl of DpnII (NEB R0543T). The reaction was incubated for 90 min at 37 °C in a thermomixer at 600 rpm and a second instance of 0.7 µl of DpnII was added followed by a second incubation of 90 min.    The nuclei were pelleted for 5 min at 1000 xg (rcf) at RT and resuspended in 135 µl of 1x CutSmart buffer (NEB B7204S). To biotinylate the free chromatin ends, dNTP-mix (NEB) (final 0.15 mM), Biotin-14-dATP (Jena Bioscience, NU-835-BIO14-S) (final 0.05 mM) and 10 U of Klenow (NEB M0210 L) were added to the reaction (final volume 150 µl) and incubated for 60 min at 25 °C. Next the free ends of the chromatin were ligated in a total volume of 1200 µl by adding ligation buffer (NEB B0202S) (1x final), 10% Triton (0.8% final), 120 µg BSA, 2000 U ligase (NEB M0202S). After two hours a second instance of 2000 U ligase was added and incubated for another 2 hours. Following the incubation, the nuclei were pelleted at 1000 g for 5 min at RT, the supernatant was discarded and the nuclei resuspended in 200 µl elution buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 1% SDS). Proteins were digested upon addition of 400 µg Proteinase K for 30 min at 55 °C. The chromatin was reverse crosslinked in the presence of 0.365 M NaCl at 68 °C overnight.   The next day DNA was purified using the ChIP DNA Clean&Concentrator Kit (Zymo Research D5205) eluted in 50 µl and biotin was removed from unligated fragments by adding NEB buffer 2 (NEB B7002S) (1x final), 12 µg of BSA, dATP and dGTP (0.025 final each), 3 U T4 DNA polymerase (NEB M0203S) in a final volume of 120 µl. The reaction was stopped by adding EDTA to a final concentration of 13 mM.  The DNA was transferred to Covaris microTUBE snap cap (520045) and sheared using the Covaris E220 (with intensifier (50014) and the following parameters duty factor 10%, peak incident power 140, cycle/burst 200, 120 sec. The DNA fragments were in the range of 200-300 bp.  Ligated and biotinylated fragments were enriched through biotin pulldown using Dynabeads MyOne Streptavidin C1 (Invitrogen 65001). The samples were filled up to a total of 400 µl with 2x binding buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 2 M NaCl) (1x final) and beads (equilibrated with TWB (5 mM Tris-HCl [pH 8], 0.5 mM EDTA, 1 M NaCl, 0.05% Tween) resuspended in 1x binding buffer were added. The beads were washed twice for 2 min at 55 °C with TWB and twice with EB-buffer (Qiagen). Last the beads were resuspended in 50 µl of EB-buffer and directly used for library preparation using the NEB Ultra II DNA Library Prep Kit for Illumina (E7645S and E6440 or E7335, E7500, E7710, E7730) following the manufacturer’s instructions except that the adapter ligation happened on the biotin beads. The fragments were released from the beads before the PCR reaction (by heating to 98 °C for 10 min) after unligated adapters had been removed. To amplify the libraries 12-13 cycles of PCR were performed.
We used NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturer’s instructions except that the adapter ligation happened on the biotin beads.

treatment_protocol: In order to deplete HP1 mRNA in S2 cells, oligos were designed (http://www.dkfz.de/signaling/e-rnai3/) that allowed the production of T7 binding sites coupled 150 bp long PCR products including the coding sequence of the third or the fourth exon of the HP1 transcript. Two independent regions were chosen to control for off-target effects. The regions were amplified from cDNA of 0-3 hr embryos. As a control a 300 bp region of GST was amplified from the vector pET41a. The PCR product was gel-purified and 500 ng -1 µg were used for in-vitro transcription using the HiScribe T7 In Vitro Transcription Kit (NEB E2050) running the reaction overnight. The fragments were purified using the MEGAclear transcription clean-up kit (Invitrogen AM1908). The RNA was eluted twice with EB-buffer heated to 95 °C. The RNA was aliquoted and stored at -80 °C.   To treat S2 cells with the dsRNA, cells were seeded in Express Five SFM (Gibco 10486025) supplemented with GlutaMAX (Gibco 35050061) at 1 Mio cells/ml in 6 ml shaking flasks. The dsRNA was thawed on ice and heated to 95 °C for 5 min, then the heating block was switched of the dsRNA was allowed to cool down to 40 °C before it was transferred to a metal rack at RT for 10 min and then transferred to ice. The dsRNA was diluted in 250 µl of Express Five SFM to reach a final amount of 60 µg of dsRNA per sample (10 µg of dsRNA in 1 Mio cells). To increase transfection efficiency 15 µl of Lipofectamine RNAiMAX (Invitrogen 13778100 ) were also diluted in 250 µl of Express Five SFM. Following the Lipofectamine mix and the diluted dsRNA were mixed and incubated for 15 min at RT. The Lipofectamine-dsRNA mix was added to the cells. The cells were grown shaking for 2 days, when a boost of dsRNA (5 µg/1 Mio cells) was added. After 4 days the cells had reached a density of 5-6 Mio cells/ml and knockdown efficiency was assessed by Western Blot and qPCR. To perform HiC the cells were harvested by centrifugation at 1300 xg for 5 min, washed with buffer A (supplemented with 10 mM of sodiumbutyrate) and fixed for 10 min at RT by adding 1 ml of 1% PFA diluted in buffer A (supplemented with 10 mM of sodiumbutyrate). The fixation was stopped by adding 100 µl of 2.5 M glycine and the cells were immediately pelleted 1300 xg for 5 min. The cells were washed again twice with buffer A (supplemented with 10 mM of sodiumbutyrate), shock frozen and stored until further use at -80 °C.

tag: clone

value: WT S2 cell line

value: S2 cell

value: RNAi HP1 dsRNA 2


title: 46-SRSF1 KD_UN_replicate 1

value: no treatment


title: HiC HP1 KD rnai2 S2 cells repl 1

description: HiC HP1 KD rnai2 S2 cells repl 1

source_name: HiC HP1 KD rnai2 S2 cells repl 1


title: 45-SRSF1 KD_SAL_replicate 3


title: ChIP-nexus_TBP_Control_TRI40min_rep2

value: Control + Triptolide 40 min


title: 49-SRSF2 KD_SAL_replicate 1

value: SRSF2 KD shRNA Knockdown


title: HiC HP1 KD rnai1 S2 cells repl 2

description: HiC HP1 KD rnai1 S2 cells repl 2

source_name: HiC HP1 KD rnai1 S2 cells repl 2

value: RNAi HP1 dsRNA 1


title: ChIP-nexus_TBP_Control_rep2

value: Control + no drug

value: Anti-TBP (Abcam, ab28175)


title: ChIP-nexus_TBP_Control_TRI40min_rep1


title: ChIP HP1 Input repl2 Covance

description:
ChIP HP1 Input repl2 Covance
HP1_covance_deepnorm_log2_smooth.bw

source_name: ChIP HP1 Input repl2 Covance

value: WT embryo at stage 5

value: embryo (Bloomington #36303)


title: 43-SRSF1 KD_SAL_replicate 1

value: SRSF1 KD shRNA Knockdown


title: HiC WT S2 cells repl 1

description: HiC WT S2 cells repl 1

source_name: HiC WT S2 cells repl 1

value: control RNAi GST


title: HiC K9M stage 5 repl2

description:
HiC K9M stage 5 repl2
K9M_all_10000_iced.matrix

source_name: HiC K9M stage 5 repl 2

extract_protocol:
Embryos were defrosted in ice cold HiC lysis buffer (10 mM Tris-HCl [pH 8], 10 mM NaCl, 0.2% Igepal,   1x Protease Inhibitor Cocktail (Roche, 11836170001)) on ice and immediately crashed with a pestle for 30 sec. The crashing of the embryos was checked under a microscope. The nuclei were pelleted for 5 min at 3000 g (rcf) and 4 °C. After discarding the supernatant the pellet was incubated in 50 µl 0.5% SDS for 10 min at RT. The reaction was quenched by adding 145 µl of water and 25 µl of 10% Triton X100.   To digest the chromatin 25 µl of DpnII buffer were added and 0.7 µl of DpnII (NEB R0543T). The reaction was incubated for 90 min at 37 °C in a thermomixer at 600 rpm and a second instance of 0.7 µl of DpnII was added followed by a second incubation of 90 min.    The nuclei were pelleted for 5 min at 1000 xg (rcf) and RT and resuspended in 135 µl of 1x CutSmart buffer (NEB B7204S). To biotinylate the free chromatin ends, dNTP-mix (NEB) (final 0.15 mM), Biotin-14-dATP (Jena Bioscience, NU-835-BIO14-S) (final 0.05 mM) and 10 U of Klenow (NEB M0210 L) were added to the reaction (final volume 150 µl) and incubated for 60 min at 25 °C. Next the free ends of the chromatin were ligated in a total volume of 1200 µl by adding ligation buffer (NEB B0202S) (1x final), 10% Triton (0.8% final), 120 µg BSA, 2000 U ligase (NEB M0202S). After two hours a second instance of 2000 U ligase was added and incubated for another 2 hours. Following the incubation the nuclei were pelleted at 1000 g for 5 min at RT, the supernatant was discarded and the nuclei resuspended in 200 µl elution buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 1% SDS). Proteins were digested upon addition of 400 µg Proteinase K for 30 min at 55 °C. The chromatin was reverse crosslinked in the presence of 0.365 M NaCl at 68 °C overnight.   The next day DNA was purified using the ChIP DNA Clean&Concentrator Kit (Zymo Research D5205) eluted in 50 µl and biotin was removed from unligated fragments by adding NEB buffer 2 (NEB B7002S) (1x final), 12 µg of BSA, dATP and dGTP (0.025 final each), 3 U T4 DNA polymerase (NEB M0203S) in a final volume of 120 µl. The reaction was stopped by adding EDTA to a final concentration of 13 mM.  The DNA was transferred to Covaris microTUBE snap cap (520045) and sheared using the Covaris E220 (with intensifier (50014) and the following parameters duty factor 10%, peak incident power 140, cycle/burst 200, 120 sec. The DNA fragments were in the range of 200-300 bp.  Ligated and biotinylated fragments were enriched through biotin pulldown using Dynabeads MyOne Streptavidin C1 (Invitrogen 65001). The samples were filled up to a total of 400 µl with 2x binding buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA, 2 M NaCl) (1x final) and beads (equilibrated with TWB (5 mM Tris-HCl [pH 8], 0.5 mM EDTA, 1 M NaCl, 0.05% Tween) resuspended in 1x binding buffer were added. The beads were washed twice for 2 min at 55 °C with TWB and twice with EB-buffer (Qiagen). Last the beads were resuspended in 50 µl of EB-buffer and directly used for library preparation using the NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturer’s instructions except that the adapter ligation happened on the biotin beads. The fragments were released from the beads before the PCR reaction (by heating to 98 °C for 10 min) after unligated adapters had been removed. To amplify the libraries 12-13 cycles of PCR were performed.
We used NEB Ultra II DNA Library Prep Kit for Illumina (E7645S) following the manufacturer’s instructions except that the adapter ligation happened on the biotin beads.

treatment_protocol: In order to induce RNAi mediated silencing of certain gene products we employed the Gal4-UAS system. By crossing males of a Gal4 driver line (Bloomington #7063) to females of the fly RNAi collection TRiP1 we obtained an F1 generation depleted for HP1 in the female germ line (Bloomington #33400). These females were crossed to siblings and produced embryos that were depleted of HP1 mRNA and protein. The same driver line and strategy was used to overexpress transgenes in the female germ line and early embryos. To collect precisely time staged embryos the flies were allowed to lay eggs on apple juice agar plates in cages for one hour at 25 °C. In order to synchronize the laying of the flies, plates were changeds for three consecutive times and the embryos were discarded. After three pre-lays, the embryos were collected for one hours and aged for another two hours at 25 °C to reach the stage of zygotic genome activation. The plate with the embryos was removed from the incubator and the embryos were dechorionated with 50% bleach, washed with water and carefully dried. The embryos were transferred in 7 ml of heptane and 5 ml of freshly prepared fixative were added. The fixative consists of  bufferof buffer A (60 mM KCl, 15 mM NaCl, 15 mM HEPES [pH 7.6], 4 mM MgCl2) and 1.8% of formaldehyde. During the formaldehyde fixation the embryos were incubated on an orbital shaker at maximum speed for 15 min. The fixation was stopped by the addition of 225 mM glycine (final concentration). The embryos were incubated for 5 min on a wheel and washed with buffer A + 0.1% Triton. After washing, the embryos were hand sorted on a cooling station under a microscope with transmitted light. The typical clear rim and the cellular membranes that form at zygotic genome activation characterize this developmental stage. The embryos were shock frozen and stored in batches of 50-100 at -80 °C.

value: embryo

value: pUASP::H3.3K9M expression


title: ChIP-nexus_TBP_Control_rep1

value: Embryonic stem cells


title: HiC WT S2 cells repl 2

description: HiC WT S2 cells repl 2

source_name: HiC WT S2 cells repl 2


title: 42-HNRNP U KD_UN_replicate 3

value: HNRNP U KD shRNA Knockdown


title: HiC HP1 KD stage 5 repl 2

description:
HiC HP1 KD stage 5 repl 2
HP1_all_no5.6_july_2019_10000_iced.matrix

source_name: HiC HP1 KD stage 5 repl 2

value: HP1-KD (Bloomington #33400)


title: RNA-seq HP1 KD repl3

description: RNA-seq HP1 KD repl3

data_processing:
Reads were mapped to D. melanogaster (build dm6) using STAR [Dobin et al 2013], using the following options: --outSJfilterReads Unique --outFilterType BySJout --outFilterMultimapNmax 1000000 --alignSJoverhangMin 6 --alignSJDBoverhangMin 2 --outFilterMismatchNoverLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --outSAMstrandField intronMotif --outFilterIntronMotifs RemoveNoncanonicalUnannotated --seedSearchStartLmax 50 --twopassMode basic. Gene expression was quantified using qCount from QuasR package [Gaidatzis et al 2015] using the "TxDb.Dmelanogaster.UCSC.dm6.ensGene" database for gene annotation and UCSC repeatMasker table for repeats annotation
Genome_build: dm6
Supplementary_files_format_and_content: csv file containing the count table

source_name: RNA-seq HP1 KD repl3

extract_protocol:
The Trizol solution was defrosted on ice filled up to 500 µl and incubated for 5 min at room temperature.  After the addition of 100 µl chloroform and vigorous mixing, the samples were spun in a table top centrifuge at 4 °C, 12.000 g for 15 min in order to separate the organic and aqueous phase. The aqueous phase was transferred in to 300 µl of chloroform and the step the first step was repeated. Subsequently the aqueous phase was transferred into isopropanol containing 20 µg of glycogen. The RNA was precipitated at -20 °C at least overnight and pelleted by centrifugation (4 °C, 12.000 g for 60 min). The pellet was washed with 80% ethanol and resuspended in DNase-digestion buffer (2 U TurboDNase Ambion, 1x TurboDNase buffer Ambion). After digestion for 30 min at 37 °C the reaction was stopped by adding EDTA to a final concentration of 15 mM and heating the samples to 75 °C for 10 min. The RNA was quantified using Qubit RNA BR Assay kit (ThermoScientific Q10210) and integrity was assessed on agarose gels or the fragmentanalyzer (Agilent).
The Illumina TruSeq Stranded Total RNA protocol was used for library preparation for next generation sequencing.

treatment_protocol: In order to induce RNAi mediated silencing of certain gene products we employed the Gal4-UAS system. By crossing males of a Gal4 driver line (Bloomington #7063) to females of the fly RNAi collection TRiP1 we obtained an F1 generation depleted for HP1 in the female germ line (Bloomington #33400). These females were crossed to siblings and produced embryos that were depleted of HP1 mRNA and protein. Embryos were collected on apple juice agar plates. In order to hand-stage the embryos they were submerged with halocarbon oil 27 (Sigma Aldrich H8773) to allow staging. Embryos of the correct developmental stage were then transferred individually into 50 µl of Trizol (ThermoScientific, 15596026), crashed with a tissue grinder, snap-frozen and stored at -80 °C until RNA extraction.