title: KTR9DpGKT2_LB_rep B

description: This sample is of Gordonia sp. KTR9DpGKT2 mutant grown under aerobic batch conditions in LB media. It is the second of three biological replicates used in this experiment, each from separate cultures

source_name: KTR9DpGKT2 in LB rich conditions rep2


title: HMEC-1 STIFF rep4

status: Public on Jul 31 2019

type: SRA

city: Seattle

first: Effie

middle: Efthimia

last: Bastounis

state: WA

address: UW Biology Life Sciences Bldg, Room 565, 4000 15th Ave NE

department: Biology

country: USA

institute: University of Washington

library_source: transcriptomic

data_processing:
Sequencing reads were obtained in Fastq format and evaluated using FastQC v0.11.5 according to the directions on the following website: http://www.bioinformatics.babraham.ac.uk/projects/fastqc/. For sequence alignment we used HISAT2 (https://ccb.jhu.edu/software/hisat2/index.shtml) and reads were mapped to reference genome grch38 to generate .bam files.
Python script HTSEQ was then used to generate counts per read (https://htseq.readthedocs.io/en/release_0.11.1/count.html). Both HISAT2 and HTSEQ were run from the command line.
For differential gene expression analysis, we used Deseq2 R package (Bioconductor version: Release 3.8, https://bioconductor.org/packages/release/bioc/html/DESeq2.html). The thresholds that we used for identifying DEGS were: (1) DESeq2 mean normalized counts > 10; (2) padj-value < 0.05; and (3) lof2foldchange >0
Genome_build: grch38
Supplementary_files_format_and_content: text files containing number of sorted counts per gene

molecule: total RNA

organism: Homo sapiens

source_name: human dermal microvascular endothelial cells

growth_protocol:
2x10^5 cells grown on 12 mm stiff 70 kPa plan polyacrylamide hydrogels for 24 h
HMEC-1 were cultured in MCDB 131 medium (Fisher Scientific; 10372-019) supplemented with 10% FBS (GemBio; 900-108), 10 ng/mL epidermal growth factor (Sigma; E9644), 1 μg/mL hydrocortisone (Sigma; H0888), and 2 mM l-glutamine (Sigma; 56-85-9).

extract_protocol:
Cells residing for 24 h on these gels were then harvested and lyzed using the QIAshredder Kit (Qiagen; 79656). mRNA was harvested using the RNeasy Plus Micro Kit (Qiagen; 74004) and eluted in 30 μL RNAase free water. RNA concentrations were measured using the nanodrop machine and were comparable between conditions. RNA quality and quantity were confirmed via bioanalyzer analysis performed by the Stanford Protein and Nucleic Acid (PAN) Facility.
RNA libraries were prepared by the Stanford Functional Genomics Facility using the KAPA stranded RNA-seq kit with RiboErase (Kit code KK8483, Roche Cat. # 07962282001), for a fragment length of 200-300 bp. Sequencing was run on the Illumina NextSeq 500 System using the High-Output Kit with 2x75 read length. We had an average of 30 million reads per sample. We used 2 lanes with 12 samples each ensuring that from each condition 3 replicates go to lane A and the other 3 to lane B (total number of replicates N = 6).

treatment_protocol: To construct polyacrylamide hydrogels the following protocol was used. Glass-bottom plates with 24 wells (MatTek; P24G-1.5-13-F) were incubated for 1 h with 500 μL of 1 M NaOH, then rinsed with distilled water, and incubated with 500 μL of 2% 3-aminopropyltriethoxysilane (Sigma; 919-30-2) in 95% ethanol for 5 min. Following rinsing with water 500 μL of 0.5% glutaraldehyde were added to each well for 30 min. Wells were rinsed with water again and dried at 60°C. To prepare polyacrylamide hydrogels of varying stiffness, mixtures containing 3–10% acrylamide (Sigma; A4058) and 0.06–0.6% bis-acrylamide (Fisher; BP1404-250) were prepared. Specifically, 3 kPa hydrogels contained 5% acrylamide and 0.1% bis-acrylamide and 70-kPa hydrogels contained 10% acrylamide and 0.6% bis-acrylamide68. For each stiffness, two mixtures were prepared, the second of which contained 0.03% 0.1 μm–diameter fluorescent beads (Invitrogen, F8803) for TFM experiments. 0.06% APS and 0.43% TEMED were added to the solutions to initiate polymerization. First, 3.6 µL of the first mixture without the beads was added at the center of each well, capped with 12-mm untreated circular glass coverslips, and allowed to polymerize for 20 min. Then 2.4 µL of the mixture containing tracer beads was added, sandwiched again with a 12-mm untreated circular glass coverslip and allowed to polymerize for 20 min. Next, 50 mM HEPES at pH 7.5 was added to the wells, and coverslips were removed. Hydrogels were UV-sterilized for 1 h and then activated by adding 200 µL of 0.5% weight/volume heterobifunctional cross-linker Sulfo-SANPAH (ProteoChem; c1111) in 1% dimethyl sulfoxide (DMSO) and 50 mM HEPES, pH 7.5, on the upper surface of the hydrogels and exposing them to UV light for 10 min. Hydrogels were washed with 50 mM HEPES at pH 7.5 and were coated with 200 µL of 0.25 mg/ml rat tail collagen I (Sigma-Aldrich; C3867) in 50 mM HEPES at pH 7.5 overnight at room temperature. After hydrogel equilibration with cell media for 30 min at 37°C, cells were seeded to a concentration of 2x10^5 cells per well directly onto the hydrogels 24 h.

tag: tissue

value: dermal microvasculature

tag: cell type

value: dermal microvascular endothelial cells

tag: nature

value: immortalized

tag: matrix

value: STIFF


title: Patient_8_6m_CD14

type: genomic

city: Stockholm

first: Ewoud

last: Ewing

address: CMM L8:05, Karolinska University Hospital

department: Clinical Neuroscience

country: Sweden

institute: Karolinska Institutet

hyb_protocol: Bisulfite conversion using EZ DNA MethylationTM Kit from Zymo Research. Product No: D5004 with 250 ng of DNA per sample followed by llumina MethylationEPIC Beadarray hybridization via Standard Illumina Protocol.

scan_protocol: Arrays were imaged using BeadArray Reader using standard recommended Illumina HiScan.

data_processing: BMIQ normalization

molecule: genomic DNA

label_protocol: Standard Illumina Protocol.

extract_protocol: Sorting strategies listed above. DNA was prepared using QIAamp DNA Micro Kit (Qiagen)

tag: individual

tag: disease state

value: multiple sclerosis

tag: treatment

value: DMF

tag: time

value: CD14+ Monocytes


title: KTR9DpGKT2_LB_rep A

type: RNA

city: Vicksburg

first: Karl

last: Indest

address: 3909 Halls Ferry Road

institute: US Army Engineer Research and Development Center

description: This sample is of Gordonia sp. KTR9DpGKT2 mutant  grown under aerobic batch conditions in LB media. It is the first of three biological replicates used in this experiment, each from separate cultures

hyb_protocol: Three hundred and twenty-five micrograms of the labeled DNA was used for subsequent hybridization to custom Agilent 8 x 15K microarrays using the Agilent hybridization kit and following the manufacturer's protocol . The hybridized arrays were washed using the Agilent Wash Buffer Kit.

scan_protocol: Washed arrays were scanned using the Agilent Technologies, High-Resolution Microarray Scanner (Model G2505C, Agilent Technologies, Santa Clara, CA, USA)

data_processing: Microarray images were analyzed using Agilent Feature Extraction software and ArrayStar v4.0.0 (DNAstar, Madison, WI) software. Expression data were log2 transformed and statistical significance was determined using a student t-test for binary transcriptome comparisons (i.e. control vs. experimental). The P-value was set at 0.05 and only those genes induced or repressed at least two-fold were considered in this paper.  Differentially expressed transcripts were assigned to their functional classifications based on pathway enrichment analysis from the Biocyc Database Collection (https://biocyc.org/)

organism: Gordonia sp. KTR9

source_name: KTR9DpGKT2 in LB rich conditions rep1

label_protocol: The RNA was used to generate cRNA using the Low Input Quick Amp Labeling Kit (Agilent, Santa Clara, California) and ethanol precipitated following the manufacturer’s directions. At each step the quality of the RNA and cRNA was analyzed using the Agilent 2100 Bioanalyzer and Total RNA Kit (Agilent) and the concentration verified using the NanoDrop 1000 Spectrophotometer (Thermo Scientific, Waltham, MA).

growth_protocol: Triplicate cultures of Gordonia sp. KTR9 and the KTR9DpGKT2 mutant were grown in LB media at 30°C with continuous shaking at 180 rpm until late log/early stationary phase.

extract_protocol: After ~48 h, cultures were centrifuged at 8,000 x g for 10 min at 4°C and total RNA was extracted using the RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) and according to the manufacturer’s instructions for RNA cleanup with an added enzymatic lysis step and mechanical disruption step added before cleanup. Enzymatic lysis and mechanical disruption was performed by resuspending the cell pellet in 100 uL TE buffer and 200 uL lysozyme (20 mg/mL), transferring the resuspended pellet to a MP Biomedicals (Solon, OH) Lysing Matrix B tube and bead beating for 30s. The optional on column DNA digestion using the Qiagen RNase-Free DNase Kit was performed to remove DNA. RNA quality and concentration was determined by analysis with an Agilent 2100 bioanalyzer.

tag: genotype/variation

value: KTR9 mutant cured of pGKT2 plasmid


title: HMEC-1 STIFF rep3


title: Patient_19_6m_CD14


title: Lung_fibroblast_normal_TGFb_5d_rep3 [Affy]

city: Ciudad de Mexico

first: Yalbi

middle: Itzel

last: Balderas-Martinez

state: Distrito Federal

address: Calzada de Tlalpan 4502, Sección XVI, Belisario Domínguez

country: Mexico

institute: INER

description: Gene expression data from normal fibroblast

hyb_protocol: Following fragmentation, 5 ug of fragmented and labeled aRNA were hybridized for 16 hr at 45ºC (GeneChip(R) Hybridization, Wash and Stain Ki, catalog 900720, Affymetrix) to GeneChip(TM) PrimeView Human Gene Expression Array ( catalog 901837, Affymetrix). GeneChips were washed and stained in the Affymetrix GeneChips(R) Fluidics Station 450.

scan_protocol: GeneChips were scanned using the Affymetrix GeneChip(TM) Scanner 3000 7G.

data_processing: The array was processed using R 3.4.2 (http://www.r-project.org/), using Bioconductor 3.6 (http://www.bioconductor.org/) with affy package. Background correction and normalization were performed using RMA.

label: biotin

source_name: normal, TGFb5d

label_protocol: Biotinylated aRNA were prepared according to the standard Affymetrix protocol from 500 ng total RNA (GeneChip(R) 3'IVT Express Kit, technical manual, catalog 901228, Affymetrix).

growth_protocol: All primary cultures were grown in Ham’s F-12 medium with 10% FBS at 37 °C in an atmosphere of 95% air and 5% CO2 until early confluence

extract_protocol: Trizol extraction of total RNA was performed according to the manufacturer's instructions.

treatment_protocol: Cells were treated with TGF-β1 (10 ng/ml), using two different time periods (24 hours or 5 days) all of them cultured in HAM´s F-12 with low serum (0.1%).

value: Lung

value: normal


title: Lung_fibroblast_normal_TGFb_24h_rep3 [Affy]

source_name: normal, TGFb24h


title: Lung_fibroblast_normal_TGFb_5d_rep2 [Affy]


title: Lung_fibroblast_normal_TGFb_5d_rep1 [Affy]


title: Patient_5_6m_CD4

value: CD4+ Tcells


title: HUVEC STIFF rep6

source_name: human umbilical vein endothelial cells

growth_protocol:
2x10^5 cells grown on 12 mm stiff 70 kPa plan polyacrylamide hydrogels for 24 h
HUVEC (Lonza C2517A) were cultured according to the manufacturer’s instructions (EGM Bullet Kit-2, Lonza CC-3162).

value: umbilical vein

value: umbilical vein endothelial cells

value: primary


title: Patient_10_6m_CD14


title: HMEC-1 STIFF rep2


title: HUVEC STIFF rep5


title: Lung_fibroblast_normal_TGFb_24h_rep1 [Affy]


title: HUVEC STIFF rep4


title: Lung_fibroblast_normal_TGFb_24h_rep2 [Affy]


title: HUVEC STIFF rep3


title: Patient_9_3m_CD14


title: Lung_fibroblast_normal_control_rep3 [Affy]

source_name: normal, control


title: HUVEC STIFF rep2


title: Patient_8_3m_CD14


title: Lung_fibroblast_normal_control_rep1 [Affy]


title: Liver fibroblasts from patient 3 without NAFLD

city: guangzhou

first: jiayu

last: liu

address: NO.74 2nd Zhongshan Road

country: China

institute: Sun Yat-Sen University

description: Primary human liver fibroblasts were isolated from liver biopsy or surgical resected samples from patients.

hyb_protocol: The labelled cirRNAs were hybridized onto Arraystar Human circRNA Arrays V2 (8×15K, Arraystar), and incubated for 17 hr at 65°C in an Agilent Hybridization Oven.

scan_protocol: Hybridized arrays were washed and scanned with Agilent Scanner G2505C.

data_processing: Data was extracted using Agilent Feature Extraction software. A series of data processing including quantile normalization were performed by R software limma package. The circRNAs that at least 1 out of 2 samples containing flags in ‘‘P’’ or ‘‘M’’ (defined by GeneSpring software) were retained for differential analyses.

source_name: Normal Liver fibroblasts

label_protocol: Total RNAs were digested with RNase R (1UI/ug, RNR07250, Epicentre) to enrich circular RNA, amplified and transcribed into fluorescent cRNA by Arraystar Super RNA Labeling Kit (074301, Arraystar) according to manufacturer’s instructions.

extract_protocol: Total RNAs were extracted by TRIzol reagent. The purity and concentration of total RNAs were determined by NanoDrop ND-1000 (Thermo Fisher Scientific).

value: Normal Liver


title: Liver fibroblasts from patient 4 without NAFLD


title: HUVEC STIFF rep1


title: HMEC-1 STIFF rep1


title: Liver fibroblasts from patient 2 without NAFLD


title: Patient_18_3m_CD4


title: Liver fibroblasts from patient 1 without NAFLD


title: Patient_10_3m_CD14


title: Patient_4_BL_CD4

source_name: BL

value: control

value: BL


title: Patient_19_BL_CD4


title: Patient_12_BL_CD4


title: Liver fibroblasts from NASH cirrhosis patient3

source_name: NASH cirrhosis Liver fibroblasts

value: NASH cirrhosis Liver


title: RNA Eno3

city: Memphis

first: Daniel

middle: Lee

last: Johnson

state: Tennessee

address: 71 S Manassas

department: Molecular Informatics Core

institute: UTHSC

hyb_protocol: Hybridization was performed using GeneChip™ WT PLUS Reagent Kit (Applied Biosystems).

scan_protocol: Microarrays were scanned on a GeneChip Scanner (Affymertrix) according to GeneChip™ WT PLUS Reagent Kit (Applied Biosystems).

data_processing: Data was processed and normalized using Thermofisher Transcriptome Analysis Console

source_name: HCI-13 breast PDX_Enobosarm

label_protocol: cDNA was labelled using GeneChip™ WT PLUS Reagent Kit (Applied Biosystems).

growth_protocol: HCI-13 PDX specimens were implanted in mice (1 mm3). Once the tumors grew to 100-200 mm3, the animals were randomized.

extract_protocol: The animals were sacrificed at the end of 30 days of treatment and tumors were snap frozen. RNA was extracted using Qiagen RNA extraction kit. RNA quality was checked and RNA with RIN numbers greater than 9 were submitted to microarray.

treatment_protocol: The animals were treated orally with vehicle or 10 mg/kg enobosarm (100 ul). Tumor volume was measured thrice weekly.

tag: cell line

value: Patient-derived xenograft (PDX) HCI-13

tag: host strain

value: NOD SCID Gamma mice

value: Enobosarm


title: RNA Eno2


title: Lung_fibroblast_normal_control_rep2 [Affy]


title: Liver fibroblasts from NASH cirrhosis patient2


title: Liver fibroblasts from NASH cirrhosis patient1


title: Liver fibroblasts from NASH cirrhosis patient4


title: Patient_9_6m_CD4

value: multiple sclerosis


title: RNA Veh3

source_name: HCI-13 breast PDX_vehicle

value: Vehicle


title: axon shPum2 rep2

status: Public on Jul 30 2019

city: New York

first: Ulrich

last: Hengst

state: NY

address: 650 West 168th Street

department: Pathology and Cell Biology

institute: Columbia University

description: processed data file: axon_shControl_vs_shPum2.txt

data_processing:
Basecalls performed by HiSeq Control Software.
Sequenced reads were mapped to Rn6 using kallisto.
Mapped reads were analyzed for differential expression using DESeq2.
Genome_build: Rn6 (Rnor_6.0)
Supplementary_files_format_and_content: *tsv: Tab-delimited text files include abundance counts from kallisto output.
Supplementary_files_format_and_content: *txt: Tab-delimited text files include differential expression from DESeq2 output, separately for axons and cell bodies.

organism: Rattus norvegicus

source_name: axons of DRGs

growth_protocol: Rat embryonic dorsal root ganglion neurons were grown in microfluidic chambers for 7 days.

extract_protocol:
Total RNA was extracted from the somatic and axonal compartments of microfluidic chambers using the Single Cell RNA Purification Kit from Norgen (Cat. 51800) as per manufacturer's instructions.
Libraries were prepared using standard or ultra-low input kits for somatic and axonal samples respectively (GENEWIZ, South Plainfield, NJ).

treatment_protocol: Neurons were transduced with lentiviruses containing shControl or shPum2 on the day of plating.

tag: developmental stage

value: axonal compartment of dorsal root ganglion neurons

tag: shRNA


title: axon shPum2 rep1


title: RNA Eno4


title: soma shPum2 rep2

description: processed data file: soma_shControl_vs_shPum2.txt

source_name: cell bodies and axons of DRGs

value: somatic compartment of dorsal root ganglion neurons


title: RNA Eno1


title: Patient_8_6m_CD4

value: DMF


title: Patient_17_3m_CD4


title: RNA Veh1


title: Patient_2_3m_CD14

value: CD14+ Monocytes


title: soma shPum2 rep1


title: axon shControl rep1

value: shControl


title: Patient_12_BL_CD14


title: soma shControl rep2


title: soma shControl rep1


title: Patient_7_6m_CD4

value: CD4+ Tcells


title: Patient_3_6m_CD4


title: Patient_3_3m_CD4


title: HS Rep 3 RNA-seq

status: Public on Jul 29 2019

city: San Deigo

first: Amy

last: Pasquinelli

state: CA

address: 9500 Gilman Drive

department: Biological Sciences

institute: UCSD

data_processing:
Reads were aligned with STAR to version WS235 of the genome using the ensembl GTF annotation file (88)
Reads were counted with FeatureCounts
Differential Expresion was determined using DESeq2
Supplementary_files_format_and_content: CSV; Contains DESeq2 output
Supplementary_files_format_and_content: WIG

organism: Caenorhabditis elegans

source_name: whole organism

growth_protocol: WT Caenorhabditis elegans (N2 Bristol) animals were cultured on OP50 bacteria at 20°C, and collected at the last larval. Standard worm synchronization methods were used.

extract_protocol:
RNA was extracted with Trizol and DNAse treated.
These samples were prepared for sequencing by rRNA depletion with Ribo-Zero rRNA Removal Mix – Gold (Illumina), and the TruSeq Stranded Total RNA Library Prep Kit (Illumina) according to the Low Sample Protocol.

treatment_protocol: The experimental Group was treated with a 4hr HS of a Temparture of 35ºC

tag: Stage

tag: strain

value: wildtype N2

value: 4 hr of 35C


title: HS Rep 1 RNA-seq


title: HS Rep 2 RNA-seq


title: Patient_10_BL_CD14


title: CTRL Rep 3 RNA-seq


title: CTRL Rep 2 RNA-seq


title: CTRL Rep 1 RNA-seq


title: Sample8_Swift_HiSeqX

city: Singapore

first: LI

last: ZHOU

address: 11 Mandalay Road

country: Singapore

institute: Lee Kong Chian School of Medicine, Nanyang Technological University

description: library prepared by Swift Biosciences Accel-NGS method, and sequenced on Illumina HiSeq X machine

library_source: genomic

data_processing:
Illumina software Real-Time Analysis v2.7.7 used for base calling. The BCL (base calls) binary is converted into FASTQ utilizing illumina bcl2fastq v2.20.
Sequenced reads were trimmed for adaptor sequence and low-quality bases using Trim Galore v0.4.4_dev and cutadapt v1.15. TruSeq: --clip_R1 8 --clip_R2 8 --three_prime_clip_R1 8 --three_prime_clip_R2 8; Swift: --clip_R2 18 --three_prime_clip_R1 18; QIAseq: --clip_R1 10 --clip_R2 10.
Trimmed reads were mapped against bisulfite-converted human genome (hg19) by using bismark. The mapping parameters were: bismark –bowtie2 -p 4 –bam --score_min L,0,-0.2.
The mapped reads were deduplicated by deduplicate_bismark and sorted by samtools v1.3 for further analysis
DNA methylated sites were identified, extracted and counted by bismark_methylation_extractor. Custom R scripts.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include methylation percentage at each CpG site generated by using bismark.

extract_protocol:
genomic DNA was extracted according to the respective sequencing providers’ usual practice.
DNA was bisulfite converted with the EZ DNA Methylation Gold Kit (Zymo Research) for use with the Swift and TruSeq library preparation methods, while the EpiTect Fast bisulfite conversion kit (QIAGEN) was to be used with the QIAseq method according to manufacturers’ preferences. The libraries were subsequently generated according to their respective protocols.

value: white blood cells

tag: library preparation method

value: Swift


title: Sample8_TruSeq_HiSeqX

description: library prepared by Illumina TruSeq  method, and sequenced on Illumina HiSeq X machine

value: TruSeq


title: Patient_15_3m_CD4


title: Sample8_QIAseq_HiSeqX

description: library prepared by QIAGEN QIAseq method, and sequenced on Illumina HiSeq X machine

value: QIAseq


title: RNA Veh2

value: Patient-derived xenograft (PDX) HCI-13


title: Sample7_Swift_HiSeqX

source_name: Neutrophils

value: Neutrophils


title: Sample7_TruSeq_HiSeqX


title: Sample7_QIAseq_HiSeqX


title: U87 MPA

city: Kyoto

first: Risa

last: Kawaguchi

state: Kyoto

address: Shogoin 53

department: Center for iPS Cell Research and Application

country: Japan

institute: Kyoto University

description:
U87 gene expression with MPA treatment
U87_MPA_(PrimeView).rma-Signal

hyb_protocol: The target was hybridized to Affymetrix Genechip Primeview Human Gene Expression Array.

scan_protocol: GeneChips were scanned using the Affemetrix Genechip Command Console software (Thermo Fisher Scientific).

data_processing: The data were analyzed with the GeneSpring GX12 Analysis Software (Agilent Technologies) using the default analysis settings.

label_protocol: The Genechip Primeview Target Synthesis and Labeling Assay were used to create biotin-labeled target cDNA.

growth_protocol: U87MG cells (ATCC) were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS).

extract_protocol: Total RNA was extracted with ISOGEN (Nippon gene) according to the manufacturer instruction.

treatment_protocol: Cells were treated with 10 μM (mycophenolic acid) MPA and/or 100 μM guanosine for 24 hr.

value: 10 μM mycophenolic acid


title: U87 MPA + Guanosine

description:
U87 gene expression with MPA and guanosine treatment
U87_MPA_+_Guanosine_(PrimeView).rma-Signal

value: 10 μM mycophenolic acid + 100 μM guanosine for 24 hr


title: U87 control

description:
Control U87 gene expression
U87_-_2_(PrimeView).rma-Signal

value: DMSO (control)


title: Patient_8_BL_CD4

value: multiple sclerosis

value: control

value: BL


title: Patient_3_6m_CD14

value: DMF

value: CD14+ Monocytes


title: Sample6_TruSeq_HiSeqX


title: Sample6_Swift_HiSeqX


title: U87 Guanosine

description:
U87 gene expression with guanosine treatment
U87_Guanosine_(PrimeView).rma-Signal

value: 100 μM guanosine for 24 hr


title: Sample6_QIAseq_HiSeqX


title: Sample5_Swift_HiSeqX


title: Sample4_TruSeq_HiSeqX_rep1

source_name: whole blood

value: whole blood


title: Sample4_Swift_HiSeqX_rep1


title: Sample4_Swift_HiSeqX_rep2


title: Sample4_TruSeq_HiSeqX_rep2


title: Patient_2_BL_CD14


title: Patient_8_BL_CD14


title: Sample3_TruSeq_HiSeqX_rep2


title: Sample3_Swift_HiSeqX_rep2


title: Sample3_QIAseq_HiSeqX


title: Sample2_TruSeq_HiSeqX_rep2


title: Sample2_QIAseq_HiSeqX


title: Sample3_TruSeq_HiSeqX_rep1


title: Sample2_Swift_HiSeqX_rep2


title: axon shControl rep2

value: axonal compartment of dorsal root ganglion neurons

value: shControl


title: Sample5_TruSeq_HiSeqX

value: white blood cells


title: Sample2_TruSeq_HiSeqX_rep1

value: TruSeq


title: Sample3_Swift_HiSeqX_rep1

value: Swift


title: Sample1_TruSeq_HiSeqX


title: Sample5_QIAseq_HiSeqX

value: QIAseq


title: Sample8_Swift_NovaSeq

description: library prepared by Swift Biosciences Accel-NGS method, and sequenced on Illumina NovaSeq machine


title: Sample7_Swift_NovaSeq

value: Neutrophils


title: Sample4_QIAseq_HiSeqX


title: Sample6_Swift_NovaSeq


title: Sample5_Swift_NovaSeq


title: Sample4_Swift_NovaSeq_rep2


title: Sample2_Swift_HiSeqX_rep1


title: Sample3_Swift_NovaSeq_rep1


title: Sample1_Swift_NovaSeq


title: Sample2_Swift_NovaSeq_rep1


title: Sample3_Swift_NovaSeq_rep2


title: H3K4me1.d2.FVFp.r1

status: Public on Jul 26 2019

city: Planegg-Martinsried

first: Filippo M.

last: Cernilogar

address: Grosshaderner Strasse 9

department: Biomedical Center

country: Germany

institute: LMU Munich

description: ES cells were seeded without feedeers in gelatin-coated plates and grown for 48h in ES standard medium (LIF/serum) supplemented with 40ng/ml doxycycline.

data_processing:
reads were mapped to the mouse genome (mm10) using bowtie (Langmead et al., 2009)
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files were generated from Homer tag directories using makeBigWig.pl

organism: Mus musculus

source_name: ES cells

extract_protocol:
1-2 million FACS-sorted cross-linked cells (1% formaldehyde, 10min RT) were lysed in 100 ul Buffer-B (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1%SDS, 1x protease inhibitors -Roche) and sonicated in a microtube (Covaris; 520045) using a Covaris S220 device until most of the DNA fragments were 200-500 base pairs long (settings: temperature 4°C, duty cycle 2%, peak incident power 105 Watts, cycles per burst 200). After shearing, lysates were centrifuged 10min, 4°C, 12000g and supernatant diluted with 900ul of Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors-Roche). 150 ul of sonicated chromatin was then incubated 4h at 4°C on a rotating wheel with 3 µg of antibody conjugated to 10 µl of magnetic beads (Dynabeads, Life Technology). Beads were washed four times with Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors-Roche) and once with Buffer-C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were re-suspended in 100µl elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) and incubated 20min at 65 °C.  Supernatant was transferred to a new tube. Crosslink reversal of immunoprecipitated DNA was carried out overnight at 65°C. Then 100µl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) was added, RNA was degraded by 4μl RNase A (10mg/ml) for 1 hour at 37°C and proteins were digested with 4μl Proteinase K (10 mg/ml) at 55°C for 2 hours. Finally, DNA was isolated by phenol:chloroform:Isoamyl alcohol purification followed by ethanol precipitation. Purified DNA was used as input for library preparation.
MicroPlex Library Preparation Kit v2 (Diagenode, cat. C05010012)

value: doxycycline (Dox) inducible Foxa2-Venus ES cell line (T119)

tag: facs sorting markers

value: Venus-positive

tag: chip antibody

value: anti-H3K4me1 (Diagenode; Pab-037-050)


title: Sample1_Swift_HiSeqX

tag: cell type

tag: tissue


title: H3K27ac.d2.FVFp.r1

value: anti-H3K27ac (Diagenode; Pab-174-050)


title: Sample1_QIAseq_HiSeqX


title: MeDIP.5mC.d3F

data_processing:
Reads were mapped to the mouse genome (mm10) using bowtie (Langmead et al., 2009).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: bigWig files were generated from Homer tag directories using makeBigWig.pl.

source_name: mesendoderm cell

growth_protocol: The cells were cultured for few consecutive passages on gelatine and ES medium. On the day of differentiation, mESCs were seeded on 10 cm gelatine coated dishes directly in endoderm differentiation medium (EDM) supplemented with 1 ng/ml of murine Wnt3a and 10 ng/ml of Activin A. Freshly prepared EDM supplemented with Wnt3a and Activin A was added every day. Cells were collected on day0, day3 and day5 for FACS isolation.

extract_protocol:
Genomic DNA from FACS-sorted cells was randomly sheared to 100-500 bp in a microtube (Covaris; 520045) using a Covaris S220 device (400 sec; 4°C; PP 140; DF 10; CB 200). Sonicated DNA was end-repaired, A-tailed, and ligated to Illumina multiplex adaptors according to NEBNext DNA library prep kit (NEB E6040S). Ligated DNA was purified using Agencourt AMPure XP beads (Beckman Coulter). 1 µg of adaptor-ligated DNA was used for each immunoprecipitation and heat-denatured at 95°C for 10min, rapidly cooled on ice and immunoprecipitated overnight at 4 ˚C with rocking agitation in 500 ml immunoprecipitation buffer (10mM sodium phosphate buffer, pH 7.0, 140mM NaCl, 0.05% Triton X-100) using 1 µl of mouse monoclonal anti-5-methylcytosine antibody (Eurogentec BI-MECY-0100) or 0.5 µl of rabbit 5-Hydroxymethylcytosine antibody (Active motif 39769). To recover the antibody-bound DNA fragments, 50µl Protein G Dynabeads (ThermoFisher) and, only to anti-5-methylcytosine IPs, 5µl of rabbit anti-mouse IgG secondary antibody (Active Motif cat.53017) were added and incubated for an additional 2 h at 4 ˚C with agitation. After immunoprecipitation, a total of seven to ten immunoprecipitation washes were performed with ice-cold immunoprecipitation buffer. Washed beads were resuspended in TE buffer with 0.25% SDS and 0.25 mg/ml proteinase K for 2 h at 55 ˚C with vigorous shaking (900 rpm). DNA was purified with the Qiagen PCR clean-up MinElute kit (Qiagen) and eluted in 30 ul. Samples were then amplified by PCR with Illumina primers (NEBNext Multiplex Oligos for Illumina cat. E7335) in a 50 μl reaction with 2× PCR master mix (NEB cat. M0541). PCR cycled as: (1) 98 °C, 30s; (2) 98 °C, 10 s; (3) 60 °C, 30 s; (4) 72 °C, 30s; (5) repeat steps (2)–(4) for 4-10 cycles; (6) 72°C, 5min. Amplified libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Quality control was carried out with a Qubit fluorometer and a Bioanalyzer (Agilent).
NEBNext DNA library prep kit (NEB E6040S).

value: double knock-in (DKI) mouse ES cell line carrying Foxa2-Venus and Sox17-Cherry gene fusions

tag: time point

tag: antibody


title: MeDIP.5mC.d0


title: MeDIP.5mC.d5FS

source_name: endoderm cells


title: d2.Foxa2n.r1

data_processing:
RNA-seq reads were mapped to the mouse genome (mm10) using STAR (Dobin et al., 2013). Expression of genes in FPKM was calculated with RSEM (Li et al., 2011).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: *_rsem.genes.results.txt: Tab-delimited text files were generated with RSEM.

growth_protocol: ES cells were seeded without feeders in gelatin-coated plates and grown for 48h in ES standard medium (LIF/serum) supplemented with 40ng/ml doxycycline.

extract_protocol:
Trizol and RNA clean and concentrator kit (Zymo research).
Smarter ultra low input-v4 (Clontech 634888) and Microplex-v2 (Diagenode C05010012).

value: doxycycline (Dox) inducible Foxa2-Venus  ES cell line (T119)

value: Venus-negative


title: MeDIP.5hmC.d3F

data_processing:
Library strategy: hMeDIP-seq
Reads were mapped to the mouse genome (mm10) using bowtie (Langmead et al., 2009).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: bigWig files were generated from Homer tag directories using makeBigWig.pl.

value: 5-Hydroxymethylcytosine (Active motif 39769)


title: d2.Foxa2n.r2


title: MeDIP.5hmC.d0


title: d3.homo.r2

growth_protocol: ES cells were split on mitomycin-treated MEFs in ESC medium without LIF. The following day, the ESCs were preplated twice to isolate the ESCs from the mitomycin-treated feeder cells and subsequently were seeded on the Wnt3a feeders cells in Endoderm Differentiation Mmedium containing Activin A (12ng/ml), refreshed every day. Cells were collected 3 days later for FACS isolation.

value: Foxa2 knock-in/knock-out (Foxa2-Venus) KO mouse ES cell line


title: d3.homo.r1


title: d2.Foxa2p.r1


title: MeDIP.5hmC.d5FS


title: d3.het.r1

value: Foxa2 knock-in/knock-out (Foxa2-Venus) control mouse ES cell line


title: d0.homo.r2

growth_protocol: ES cells were split on red-fluorescent mitomycin-treated MEFs in ESC medium without LIF. The following day were collected for FACS isolation.

value: Cherry-negative


title: Sample4_Swift_NovaSeq_rep1

value: whole blood


title: Sample2_Swift_NovaSeq_rep2


title: d0.homo.r1


title: Patient_6_6m_CD4

tag: individual

tag: disease state

value: multiple sclerosis

value: DMF

value: CD4+ Tcells


title: d3.het.r2


title: CD14_202_Sepsis

city: Collegeville

first: James

middle: Raymond

last: Brown

state: PA

address: 1250 S. Collegeville Road

institute: GlaxoSmithKline

description: 202_009A_Blood_CD14_20160629_Sepsis_Early_AR014_S22_L005_001

data_processing:
An initial quality check of raw FASTQ data was performed using FastQC.
Reads that were either less than 25bp, or had a maximum quality score below Q15, or average quality score below Q10 were discarded. The quality filtered reads were mapped to the human reference genome GRCh38/hg38 (Dec 2013) using STAR.
The read counts were called using Subread.
Differential gene expression analysis was performed for CD4+, CD8+ and CD14+ samples using DESeq2 in R Bioconductor.
Genome_build: hg38
Supplementary_files_format_and_content: tab-delimited text file includes count values for each sample

extract_protocol:
RNA was extracted and purified using Qiagen’s RNeasy Mini Kit on the QIAcube.
Starting with 350 nanograms of total RNA, barcoded libraries were prepared using Illumina’s TruSeq RNA Library Prep totalRNA kit. The quality of the libraries was confirmed using the Agilent Bioanalyzer DNA1000 Assay. The libraries were quantified and normalized by qPCR using KAPA Library Quantification Kit.

tag: patient group

value: Sepsis

tag: subjectid

tag: sepsis stage

value: Early

tag: sampleID


title: d0.het.r2

tag: cell line

tag: genotype/variation


title: d0.het.r1


title: MeDIP.Input


title: Gata4.d5FS.r1

extract_protocol:
1-2 million FACS-sorted cross-linked cells (1% formaldehyde, 10min RT) were lysed in 100 ul Buffer-B-0.3 (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0,3%SDS, 1x protease inhibitors -Roche) and sonicated in a microtube (Covaris; 520045) using a Covaris S220 device until most of the DNA fragments were 200-500 base pairs long (settings: temperature 4°C, duty cycle 2%, peak incident power 105 Watts, cycles per burst 200). After shearing, lysates were centrifuged 10min, 4°C, 12000g and supernatant diluted with 1 volume of Dilution Buffer (1mM EGTA 300 mM NaCl, 2% Triton x-100, 0.2% sodium deoxycholate, 1x protease inhibitors-Roche). Sonicated chromatin was then incubated 4h at 4°C on a rotating wheel with 6 ug of antibody conjugated to 20 µl of magnetic beads  (Dynabeads, Life Technology). Beads were washed four times with Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors) and once with Buffer-C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were then incubated with 70 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2 μl of Proteinase K (20mg/ml) for 1 hour at 55°C and 8 hours at 65°C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads for 1 minute and eluates were combined and incubated with another 1 μl of Proteinase K for 1 hour at 55°C. Finally, DNA was purified with SPRI AMPure XP beads (Beckman Coulter) (sample-to-beads ratio 1:2). Purified DNA was used as input for library preparation.
MicroPlex Library Preparation Kit v2 (Diagenode, cat. C05010012).

value: anti-Gata4 (R&D Systems; AF2606)


title: Nanog.d0.r1

value: anti-Nanog (Bethyl lab; A300-397-A)


title: Foxa2.d5FS.r1

value: anti-Foxa2 (Santa Cruz; sc6554x)


title: Foxa2.d5FS.r2


title: RNA_d5FS_r2

data_processing:
Paired-end RNA-seq reads were mapped to the mouse genome (mm10) using STAR (Dobin et al., 2013). Expression of genes in FPKM was calculated with RSEM (Li et al., 2011).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: *_rsem.genes.results.txt: Tab-delimited text files were generated with RSEM.

extract_protocol:
Total RNA was extracted with Trizol and RNA clean and concentrator kit (Zymo research), including the on-column DNase step. Ribo-depletion with Ribozero goldkit (Illumina MRZG126).
Illumina TruSeq V2 RNA prep kit.


title: Gata4.d3F.r1

source_name: mesendoderm cells


title: Foxa2.d3F.r2


title: Foxa2.d3F.r1


title: d2.Foxa2p.r2

value: Venus-positive


title: RNA_d5FS_r1

value: double knock-in (DKI) mouse ES cell line carrying Foxa2-Venus and Sox17-Cherry gene fusions


title: Foxa2.d2.FVFp.r2

value: doxycycline (Dox) inducible Foxa2-Venus ES cell line (T119)


title: RNA_d3F_r1


title: RNA_d3F_r2


title: Foxa2.d2.FVFp.r1


title: Input.d5FS.r1

extract_protocol:
1-2 million FACS-sorted cross-linked cells (1% formaldehyde, 10min RT) were lysed in 100 ul Buffer-B (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1%SDS, 1x protease inhibitors -Roche) and sonicated in a microtube (Covaris; 520045) using a Covaris S220 device until most of the DNA fragments were 200-500 base pairs long (settings: temperature 4°C, duty cycle 2%, peak incident power 105 Watts, cycles per burst 200). After shearing, lysates were centrifuged 10min, 4°C, 12000g and supernatant diluted with 900ul of Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors-Roche). 150 ul of sonicated chromatin was then incubated 4h at 4°C on a rotating wheel with 3 µg of antibody conjugated to 10 µl of magnetic beads (Dynabeads, Life Technology). Beads were washed four times with Buffer-A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors-Roche) and once with Buffer-C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were re-suspended in 100µl elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1% SDS) and incubated 20min at 65 °C. Supernatant was transferred to a new tube. Crosslink reversal of immunoprecipitated DNA was carried out overnight at 65°C. Then 100µl TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) was added, RNA was degraded by 4μl RNase A (10mg/ml) for 1 hour at 37°C and proteins were digested with 4μl Proteinase K (10 mg/ml) at 55°C for 2 hours. Finally, DNA was isolated by phenol:chloroform:Isoamyl alcohol purification followed by ethanol precipitation. Purified DNA was used as input for library preparation.
MicroPlex Library Preparation Kit v2 (Diagenode, cat. C05010012).


title: Input.d3F.r1


title: RNA_d0_r2


title: H3K4me1.d3F.r2

value: anti-H3K4me1 (Diagenode; Pab-037-050)


title: H3K4me1.d0.r2


title: H4K20me3.d5FS.r1

value: anti-H4K20me3 (Diagenode; Pab-057-050)


title: H4K20me3.d3F.r1


title: Input.d0.r1


title: H4K20me3.d0.r1


title: H3K27me3.d5FS.r2

value: anti-H3K27me3 (Diagenode; Pab-069-050)


title: H3K9me3.d5FS.r1

value: anti-H3K9me3 (Diagenode; Pab-056-050)


title: H3K9me3.d3F.r1


title: H3K9me3.d0.r1


title: H3K27ac.d5FS.r2

value: anti-H3K27ac (Diagenode; Pab-174-050)


title: H3K27me3.d3F.r2


title: H3K27me3.d0.r2


title: H3K27ac.d3F.r2


title: H3K4me3.d5FS.r1

value: anti-H3K4me3 (Diagenode; Pab-003-050)


title: H3K27ac.d0.r2


title: H3K4me1.d5FS.r1


title: H3K4me1.d3F.r1


title: H3K4me1.d0.r1


title: H3K4me3.d0.r1


title: H3K27me3.d5FS.r1


title: RNA_d0_r1


title: H3K4me1.d5FS.r2


title: H3K27me3.d0.r1


title: H3K27ac.d0.r1


title: H3K27ac.d5FS.r1


title: H3K27me3.d3F.r1


title: H3K27ac.d3F.r1


title: hs_control_8cell_3PN_SmartSeq_rep2

status: Public on Jul 05 2019

city: Beijing

first: Wei

last: Xie

state: Beijing

address: Qinghuayuan

department: School of Life Science

institute: Tsinghua University

data_processing:
The single-end CUT&RUN reads were aligned to mm9 or hg19 reference genome with random chromosome cleaned by Bowtie (version 2.2.2)(Langmead and Salzberg, 2012) under the parameters –t –q –N 1 –L 25. The paired-end CUT&RUN reads were aligned with the parameters: -t –q –N 1 –L 25 –X 1000 --no-mixed --no-discordant. All unmapped reads, non-uniquely mapped reads, reads with low mapping quality (MAPQ < 20) and PCR duplicates were removed. For downstream analysis, we normalized the read counts by computing the numbers of reads per kilobase of bin per million of reads sequenced (RPKM) for 100-bp bins of the genome. To minimize the batch and cell type variation, RPKM values across whole genome were further Z-score normalized. To visualize the CUT&RUN signals in the UCSC genome browser, we generated the RPKM values on a 100bp-window base. ATAC-seq reads were aligned to the hg19 genome using bowtie 2.2.2, replicates of same stage were pooled together and rpkm in wiggle files were counted by the number of reads falling into 100bp bin in the genome.
narrow peaks were called using MACS2 with the parameters -g hs -nomodel -nolambda and broad peaks were called using MACS2 with the parameters -g hs -broad --broad-cutoff 0.05 -nomodel -nolambda
Genome_build: mm9 or hg19
Supplementary_files_format_and_content: bedGraph formatted files including the RPKM values in 100-bp bins of the mm9 or hg19 genome

molecule: polyA RNA

source_name: embryo

extract_protocol:
RNA-Seq libraries were prepared using Smart-Seq2 protocol with slightly modification
After removing the zona pellucida, embryonic cells were washed with PBS and incubated in lysis buffer.

treatment_protocol: DMSO treated human embryos

value: embryo

tag: developmental stage

tag: cut&run-antibody


title: mm_PN5_H3K4me3_rep1

data_processing:
The single-end CUT&RUN reads were aligned to mm9 or hg19 reference genome with random chromosome cleaned by Bowtie (version 2.2.2)(Langmead and Salzberg, 2012) under the parameters –t –q –N 1 –L 25. The paired-end CUT&RUN reads were aligned with the parameters: -t –q –N 1 –L 25 –X 1000 --no-mixed --no-discordant. All unmapped reads, non-uniquely mapped reads, reads with low mapping quality (MAPQ < 20) and PCR duplicates were removed. For downstream analysis, we normalized the read counts by computing the numbers of reads per kilobase of bin per million of reads sequenced (RPKM) for 100-bp bins of the genome. To minimize the batch and cell type variation, RPKM values across whole genome were further Z-score normalized. To visualize the CUT&RUN signals in the UCSC genome browser, we generated the RPKM values on a 100bp-window base.
narrow peaks were called using MACS2 with the parameters -g hs -nomodel -nolambda and broad peaks were called using MACS2 with the parameters -g hs -broad --broad-cutoff 0.05 -nomodel -nolambda
Genome_build: mm9 or hg19
Supplementary_files_format_and_content: bedGraph formatted files including the RPKM values in 100-bp bins of the mm9 or hg19 genome

extract_protocol:
CUT&RUN was conducted following the published protocol (Skene et al., 2018) with modifications.Oocytes or embryos removed zona pellucida were transferred into 0.2mL conventional, non-low-binding PCR tube (Axygen). Cells were attached to a solid support using concanavalin A–coated magnetic beads to allow simple handling. Following in situ binding of the antibody and pA-MN specifically to the target protein, pA-MN was activated by calcium at 0 °C. Cleaved fragments released with two cuts were free to diffuse out of the nuclei. After removing the intact nuclei by centrifugation, the supernatant containing released chromatin fragments were used to extract DNA directly for library construction.
Purified DNA was subjected to Tru-seq library construction using either KAPA Hyper Prep Kit (KAPA biosystems, KK8502) or NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S).

value: PN5 zygote


title: hs_control_8cell_3PN_SmartSeq_rep1


title: mm_GV_H3K4me3_rep1

value: GV oocyte


title: mm_Late2cell_H3K4me3_rep1

value: Late 2-cell


title: hs_ICM_2PN_H3K27ac_rep2

data_processing:
Library strategy: CUT&RUN
The single-end CUT&RUN reads were aligned to mm9 or hg19 reference genome with random chromosome cleaned by Bowtie (version 2.2.2)(Langmead and Salzberg, 2012) under the parameters –t –q –N 1 –L 25. The paired-end CUT&RUN reads were aligned with the parameters: -t –q –N 1 –L 25 –X 1000 --no-mixed --no-discordant. All unmapped reads, non-uniquely mapped reads, reads with low mapping quality (MAPQ < 20) and PCR duplicates were removed. For downstream analysis, we normalized the read counts by computing the numbers of reads per kilobase of bin per million of reads sequenced (RPKM) for 100-bp bins of the genome. To minimize the batch and cell type variation, RPKM values across whole genome were further Z-score normalized. To visualize the CUT&RUN signals in the UCSC genome browser, we generated the RPKM values on a 100bp-window base. ATAC-seq reads were aligned to the hg19 genome using bowtie 2.2.2, replicates of same stage were pooled together and rpkm in wiggle files were counted by the number of reads falling into 100bp bin in the genome.
narrow peaks were called using MACS2 with the parameters -g hs -nomodel -nolambda and broad peaks were called using MACS2 with the parameters -g hs -broad --broad-cutoff 0.05 -nomodel -nolambda
Genome_build: mm9 or hg19
Supplementary_files_format_and_content: bedGraph formatted files including the RPKM values in 100-bp bins of the mm9 or hg19 genome

extract_protocol:
Purified DNA was subjected to Tru-seq library construction using either KAPA Hyper Prep Kit (KAPA biosystems, KK8502) or NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645S).
CUT&RUN was conducted following the published protocol (Skene et al., 2018) with modifications.Oocytes or embryos removed zona pellucida were transferred into 0.2mL conventional, non-low-binding PCR tube (Axygen). Cells were attached to a solid support us concanavalin A–coated magnetic beads to allow simple handling. Following in situ binding of the antibody and pA-MN specifically to the target protein, pA-MN was activated by calcium at 0 °C. Cleaved fragments released with two cuts were free to diffuse out of the nuclei. After removing the intact nuclei by centrifugation, the supernatant containing released chromatin fragments were used to extract DNA directly for library construction.

value: ICM

value: H3K27ac(Active Motif,39133)


title: hs_ICM_2PN_H3K27ac_rep1

data_processing:
Library strategy: CUT&RUN
narrow peaks were called using MACS2 with the parameters -g hs -nomodel -nolambda and broad peaks were called using MACS2 with the parameters -g hs -broad --broad-cutoff 0.05 -nomodel -nolambda
Genome_build: mm9 or hg19
Supplementary_files_format_and_content: bedGraph formatted files including the RPKM values in 100-bp bins of the mm9 or hg19 genome


title: hs_8cell_3PN_H3K27ac_rep2


title: hs_TBE_8cell_3PN_SmartSeq_rep2

treatment_protocol: α-amanitin treated human embryos


title: hs_TE_D7_2PN_H3K27me3_rep1

value: D7 TE


title: hs_TBE_8cell_3PN_SmartSeq_rep1


title: H3K4me3.d3F.r1


title: hs_8cell_3PN_H3K27ac_rep1


title: hs_ICM_D7_2PN_H3K27me3_rep1

value: D7 ICM


title: hs_TE_2PN_H3K27me3_rep2

value: TE


title: hs_TE_2PN_H3K27me3_rep1


title: hs_control_8cell_3PN_H3K4me3_rep1


title: hs_GV_ATAC_rep2

extract_protocol:
ATAC-Seq libraries were prepared using ATAC-seq developed by Buenrostro et al. with slightly modification
After removing the zona pellucida, embryonic cells were washed with PBS and incubated in lysis buffer.


title: hs_ICM_2PN_H3K4me3_rep2


title: hs_ICM_2PN_H3K27me3_rep1


title: hs_ICM_2PN_H3K4me3_rep1


title: hs_ICM_2PN_H3K27me3_rep2


title: hs_8cell_3PN_H3K27me3_rep2


title: hs_8cell_3PN_H3K27me3_rep1


title: hs_8cell_3PN_H3K4me3_rep2


title: mm_8cell_H3K4me3_rep1_seq1


title: hs_8cell_2PN_H3K27me3_rep1


title: hs_8cell_2PN_H3K4me3_rep1


title: hs_8cell_2PN_H3K4me3_rep2


title: hs_4cell_3PN_H3K27me3_rep1


title: hs_4cell_2PN_H3K4me3_rep1


title: hs_4cell_2PN_H3K4me3_rep2


title: hs_2cell_3PN_H3K27me3_rep1


title: hs_4cell_3PN_H3K4me3_rep1


title: hs_MI_H3K4me3_rep1

value: MI oocyte


title: hs_GV_H3K27me3_rep2


title: hs_2cell_3PN_H3K27me3_rep2


title: hs_GV_H3K27me3_rep1


title: hs_GV_H3K4me3_rep1


title: hs_GV_H3K4me3_rep2


title: hs_GV_ATAC_rep1


title: hs_TBE_8cell_3PN_H3K4me3_rep1


title: 2h_mature_repliATAC_S2spikein_rep1

status: Public on May 21 2019

city: Copenhagen

first: Anja

last: Groth

address: Blegdamsvej 3B

country: Denmark

institute: CPR

description:
repliATAC_spikein_5kbcentred_EnsemblTSS_RPM_+1_log2.bed
repliATAC_spikein_5kbcentred_mESCenhancers_RPM_+1_log2.bed

data_processing:
Raw reads trimmed using Trim Galore! (Galaxy version 0.4.2) to remove low-quality reads and adaptor sequence.
Libraries were mapped to the mm10 genome assembly using Bowtie2 (Galaxy version 2.2.6.2).
For repli-ATAC-seq libraries, duplicates and reads mapping to the mitochondrial genome, a mitochondrial homolog blacklist (Buenrostro et al, 2013) or the Broad Institute's blacklist (Encode Project Consortium, 2012) were discarded. Forward reads were shifted +4 bp, reverse reads were shifted -5 bp. Reads greater than 100 bp in length were discarded except where indicated.
For ChOR-seq libraries, duplicates and reads mapping to the Broad Institute's blacklist (Encode Project Consortium, 2012) were discarded. Remaining reads were extended by 250 bp for peak calling and downstream analyses.
Peak calling was done using custom R scripts.
References: Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., and Greenleaf, W.J. (2013). Transposition  of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding  proteins and nucleosome position. Nat Meth 10, 1213.; ENCODE Project Consortium (2012). An integrated encyclopedia of DNA elements in the human  genome. Nature 489, 57–74.; Whyte, W.A., Orlando, D.A., Hnisz, D., Abraham, B.J., Lin, C.Y., Kagey, M.H., Rahl, P.B., Lee,  T.I., and Young, R.A. (2013). Master transcription factors and mediator establish super-enhancers  at key cell identity genes. Cell 153, 307–319.
Genome_build: mm10
Supplementary_files_format_and_content: Processed files for the repli-ATAC-seq time series contain the mm10 coordinates for replicated peaks.
Supplementary_files_format_and_content: Processed files for the repli-ATAC-seq inhibitor libraries contain corrected, n+1, log-transformed read counts from 1 kb windows with a 500 bp step.
Supplementary_files_format_and_content: Processed files for RNA polymerase ChOR-seq contain the mm10 coordinates for replicated peaks from total ChIP.
Supplementary_files_format_and_content: Processed files for repli-ATAC-seq performed with spike-in contain corrected, n+1, log-transformed read counts from 5 kb centred on either Ensembl TSSs or mESC enhancers as defined in (Whyte et al, 2013).

source_name: Embryonic stem cells

growth_protocol: Mouse embryonic stem cells (ESCs) were grown on gelatinized plates in 15% fetal bovine serum with penicillin/ streptomycin, GlutaMAX, non-essential amino acids, beta-mercaptoethanol, and homemade LIF.

extract_protocol:
After the appropriate treatment, cells were trypsinized and collected in media. In parallel, and independently, EdU-labelled Drosophila S2 cells were trypsinized, collected in media, and 100 cells were added to each mESC repli-ATAC-seq sample. Samples were then processed and tagmented using TD buffer and TDE1 enzyme (Illumina).
EdU-positive samples were biotinylated via Click-IT (Invitrogen) and purified with Ampure XP beads (Beckman Coulter). Biotinylated products were captured using MyOne Streptavidin T1 beads (Thermo Fisher Scientific), and the unbound fraction in the supernatant from streptavidin pulldown was purified using the MinElute Reaction Cleanup kit (Qiagen). Steady state samples were purified following tagmentation using the MinElute PCR Purification Kit (Qiagen). Samples were amplified using NEBNext High-Fidelity 2x PCR Master Mix (NEB) and final libraries were purified using Ampure XP beads.

treatment_protocol: Steady state samples were untreated. EdU-positive samples were incubated in media containing EdU (20 uM final concentration) for 10 minutes, then either collected for processing (nascent samples) or cultured for the appropriate maturation interval in media containing 10 uM thymidine before processing. Inhibitor-treated samples were incubated in media with DMSO, DRB, or triptolide for 2 hours prior to EdU labelling.

tag: passages

tag: strain

tag: timepoint

value: 2 hours Mature

tag: molecule

value: EdU-labelled genomic DNA


title: Nascent_repliATAC_S2spikein_rep2

tag: cell type

value: Nascent


title: 2h_mature_ChORseq_RNApolIIS5phos_rep1

description: RNApolIIS5phos_totalChIP_replicated_peaks.bed

data_processing:
Library strategy: ChOR-seq
Raw reads trimmed using Trim Galore! (Galaxy version 0.4.2) to remove low-quality reads and adaptor sequence.
Libraries were mapped to the mm10 genome assembly using Bowtie2 (Galaxy version 2.2.6.2).
For repli-ATAC-seq libraries, duplicates and reads mapping to the mitochondrial genome, a mitochondrial homolog blacklist (Buenrostro et al, 2013) or the Broad Institute's blacklist (Encode Project Consortium, 2012) were discarded. Forward reads were shifted +4 bp, reverse reads were shifted -5 bp. Reads greater than 100 bp in length were discarded except where indicated.
For ChOR-seq libraries, duplicates and reads mapping to the Broad Institute's blacklist (Encode Project Consortium, 2012) were discarded. Remaining reads were extended by 250 bp for peak calling and downstream analyses.
Peak calling was done using custom R scripts.
References: Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y., and Greenleaf, W.J. (2013). Transposition  of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding  proteins and nucleosome position. Nat Meth 10, 1213.; ENCODE Project Consortium (2012). An integrated encyclopedia of DNA elements in the human  genome. Nature 489, 57–74.; Whyte, W.A., Orlando, D.A., Hnisz, D., Abraham, B.J., Lin, C.Y., Kagey, M.H., Rahl, P.B., Lee,  T.I., and Young, R.A. (2013). Master transcription factors and mediator establish super-enhancers  at key cell identity genes. Cell 153, 307–319.
Genome_build: mm10
Supplementary_files_format_and_content: Processed files for the repli-ATAC-seq time series contain the mm10 coordinates for replicated peaks.
Supplementary_files_format_and_content: Processed files for the repli-ATAC-seq inhibitor libraries contain corrected, n+1, log-transformed read counts from 1 kb windows with a 500 bp step.
Supplementary_files_format_and_content: Processed files for RNA polymerase ChOR-seq contain the mm10 coordinates for replicated peaks from total ChIP.
Supplementary_files_format_and_content: Processed files for repli-ATAC-seq performed with spike-in contain corrected, n+1, log-transformed read counts from 5 kb centred on either Ensembl TSSs or mESC enhancers as defined in (Whyte et al, 2013).

extract_protocol:
Fixed cells were lysed for 20 minutes in ice-cold lysis buffer (100mM NaCl, 66mM Tris-HCl pH 8, 5mM EDTA, 0.3% SDS, 1.6% Triton X-100) supplemented with protease inhibitors, passed through a 21G needle, and sonicated using a Covaris M220 (Thermo Fisher) to obtain chromatin fragment sizes of 250-500 bp. Sonicated chromatin was centrifuged at 14,000 rpm at 4ºC for 10 minutes and the supernatant used for subsequent steps. In parallel, and independently, Drosophila S2 cells were fixed, lysed and sonicated as described above. After sonication, mESC input chromatin required for the analysis of all time-points from the same time course experiment was mixed with Drosophila S2 chromatin (2.5% of total chromatin). Chromatin was immunoprecipitated by incubating sonicated chromatin with antibody (Abcam, ab5131).
Libraries were constructed using the KAPA Hyperprep kit following manufacturer’s instructions, except that 1.25 µM of Illumina-compatible indexed adapters (Pentabase) were ligated to A-tailed DNA. Following adapter ligation, biotin-TEG-azide (Berry&Associates) was used to isolate EdU-labelled DNA via Click-iT (Invitrogen). Biotinylated products were captured using MyOne Streptavidin T1 beads (Thermo Fisher Scientific) and subjected to PCR amplification following the KAPA Hyperprep kit protocol.

tag: chip antibody

value: RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody - ChIP Grade (Abcam, ab5131)


title: RNApolIIS5phos_ChIP_rep2

value: genomic DNA


title: RNApolIIS5phos_ChIP_rep1


title: 1h_mature_ChORseq_RNApolIIS5phos_rep2

value: 1 hour Mature


title: hs_8cell_3PN_H3K4me3_rep1

tag: antibody


title: 2h_mature_ChORseq_RNApolIIS5phos_rep2


title: 1h_mature_ChORseq_RNApolIIS5phos_rep1


title: 30m_mature_ChORseq_RNApolIIS5phos_rep1

value: 30 minutes Mature


title: 2h_Mature_triptolide_unbound_rep3

description: repliATAC_unbound_inhibitor_1kbwindow_500bpstep_RPM_+1_log2.txt.gz

extract_protocol:
After the appropriate treatment, cells were trypsinized and collected in media. Samples were then processed and tagmented using TD buffer and TDE1 enzyme (Illumina).
EdU-positive samples were biotinylated via Click-IT (Invitrogen) and purified with Ampure XP beads (Beckman Coulter). Biotinylated products were captured using MyOne Streptavidin T1 beads (Thermo Fisher Scientific), and the unbound fraction in the supernatant from streptavidin pulldown was purified using the MinElute Reaction Cleanup kit (Qiagen). Steady state samples were purified following tagmentation using the MinElute PCR Purification Kit (Qiagen). Samples were amplified using NEBNext High-Fidelity 2x PCR Master Mix (NEB) and final libraries were purified using Ampure XP beads.

tag: treatment

value: Triptolide


title: Nascent_ChORseq_RNApolIIS5phos_rep1


title: 2h_Mature_DMSO_unbound_rep2

value: DMSO control


title: 2h_Mature_DRB_unbound_rep1

value: DRB


title: 2h_Mature_DRB_unbound_rep2


title: 2h_Mature_DMSO_unbound_rep3


title: 2h_Mature_DRB_unbound_rep3


title: 2h_mature_repliATAC_S2spikein_rep2


title: CS_seedlings_H3K9me2_ChIP_seq_rep2

status: Public on Jun 21 2019

city: shanghai

first: yijing

last: zhang

address: 2005 Songhu Road

department: Biochemistry

institute: Fudan University

data_processing:
Sequencing reads were cleaned with Trim Galore v0.4.4, Trimmomatic v0.36 and sickle, including removing bases with low quality score (<25) and irregular GC content, and cutting sequencing adaptors followed by filtering short reads.
The cleaned reads were mapped to International Wheat Genome Sequencing Consortium (IWGSC) RefSeq v1.0 using BWA 0.7.5a-r405  for ChIP-seq and DNaseI-seq  data,  HISAT2 2.1.020 for RNA-seq data ,  Bismark_v0.19.0 for bisulfite-seq data , all with default settings.
For ChIP-seq  and DNaseI-seq data, MACS1.3.7 was used to identify read-enriched regions (peaks) with combined criteria: P value < 1e–50 and fold-change >32.  ChIP-seq data Target genes were defined as genes with a peak within or nearby the gene body (±2 kb).
Genome_build: IWGSC RefSeq v1.0
Supplementary_files_format_and_content: In ChIP-seq, peak files were provided; In RNA-seq, read counts for genes (IWGSC RefSeq v1.0) were provided;In DNaseI-seq, peak files were provided; In Bisulfite-seq, BigWig files with position and  methylation ratio were provided.

organism: Triticum aestivum

source_name: Triticum aestivum 12-days-old seedlings

growth_protocol: 16h light, 8h dark, 22°C

extract_protocol:
Chromatin or RNA was extracted from 12-days-old seedlingss, using standard protocols. 2 microgram mRNA, 2 microgram total RNA (rRNA depleted) were used to prepare mRNA-seq and lncRNA-seq. 2.2 microgram DNA extracted from the harvested seedlings were used to prepare ChIP-seq DnaseI-seq and Bisulfite-seq   .
Library construction and deep sequencing were performed by Genergy Biotechnology Co. Ltd. (Shanghai, China) using Illumina HiSeq 3000 following the manufacturer’s instructions  (Illumina) to produce 150bp paired-end reads.

treatment_protocol: No treatment applied.

tag: cultivar

value: Chinese Spring

tag: tissue

value: 12-days-old seedlings

tag: chip antibody manufacturer

value: Abcam

tag: chip antibody catalog #


title: 2h_Mature_triptolide_unbound_rep1


title: Nascent_triptolide_unbound_rep1


title: Nascent_triptolide_unbound_rep3


title: Nascent_triptolide_unbound_rep2


title: Nascent_DRB_unbound_rep1


title: Nascent_repliATAC_S2spikein_rep1


title: Nascent_DMSO_unbound_rep3


title: Nascent_DRB_unbound_rep2


title: Nascent_DRB_unbound_rep3


title: 30m_mature_ChORseq_RNApolIIS5phos_rep2


title: 2h_Mature_triptolide_unbound_rep2


title: Nascent_DMSO_unbound_rep1


title: 2h_Mature_DRB_repliATAC_rep1


title: 2h_Mature_DMSO_repliATAC_rep3


title: 2h_Mature_DRB_repliATAC_rep2


title: 2h_Mature_DRB_repliATAC_rep3


title: 2h_Mature_triptolide_repliATAC_rep1


title: 2h_Mature_DMSO_repliATAC_rep2


title: 2h_Mature_DMSO_repliATAC_rep1


title: Nascent_triptolide_repliATAC_rep3


title: Nascent_DRB_repliATAC_rep1


title: Nascent_triptolide_repliATAC_rep1


title: Nascent_DRB_repliATAC_rep2


title: Nascent_DRB_repliATAC_rep3


title: Nascent_DMSO_repliATAC_rep3


title: Nascent_DMSO_unbound_rep2


title: Nascent_DMSO_repliATAC_rep1


title: Steady_state_rep2


title: 2h_Mature_repliATAC_rep2


title: 2h_Mature_repliATAC_rep3


title: Steady_state_rep1


title: Nascent_DMSO_repliATAC_rep2


title: 2h_Mature_repliATAC_rep1


title: 1h_Mature_repliATAC_rep3


title: 2h_Mature_triptolide_repliATAC_rep3


title: 1h_Mature_repliATAC_rep2


title: 1h_Mature_repliATAC_rep1


title: Nascent_triptolide_repliATAC_rep2


title: 30m_Mature_repliATAC_rep3


title: Nascent_repliATAC_rep1


title: 30m_Mature_repliATAC_rep2


title: Nascent_repliATAC_rep3


title: PRO-seq SNF5 rep3

status: Public on Apr 12 2019

city: Nashville

first: Jing

last: Wang

state: TN

address: 2220 Pierce Avenue

institute: Vanderbilt University Medical Center

description:
nuclear RNA
PROseq-pp_gb_counts.txt

data_processing:
After adapter trimming and low quality sequence removal by cutadapt, PRO-seq reads longer than 15bp were reversed complemented using FastX tools.
Reverse complements of the trimmed reads were aligned to the human genome hg19 using Bowtie2.
Reads mapped to rRNA loci and reads with mapping quality less than 10 were removed.
Bam files were given to NRSA (http://bioinfo.vanderbilt.edu/NRSA/) to estimate RNA polymerase abundance in proximal-promoter and gene body regions of genes, to calculate pausing index and pausing index alterations, and to detect enhancers and quantify eRNA changes.
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie2.
Peaks were called by MACS2 with a q-value of 0.01.
Differential binding peaks were identified using DiffBind based on consensus peaks occurring at least two samples.
Adapter sequences of ATAC-seq reads were trimmed by cutadapt (cutadapt -a CTGTCTCTTA  TACACATCT-minimum-length 15 -paired-output), then aligned to the human genome hg38 using Bowtie2 (bowtie2 -p 8 -X 2000 -q --no-mixed --no-discordant).
Peaks were called using MACS2 with q-value of 0.001 (callpeak -q 0.001 -nomodel -extsize 140).
Peaks were annotated and enriched motifs were identified by Homer.
Differential enriched peaks were identified using DiffBind based on consensus peaks occurring at least two samples.
genome build: hg19, hg38
processed data files format and content: ChIP-seq: one peak file per Sample
processed data files format and content: ATAC-seq: one peak file per Sample
processed data files format and content: PRO-seq: tab-delimited text file include raw counts for proximal-promoter and gene body region, and density for gene body

source_name: MRT cells

growth_protocol: G401 cells  were routinely grown in DMEM  with 10% FBS and 1%Penicillin/Streptomycin. To express TET-inducible constructs G401 were plated at a low density and transduced for 2 days with lentiviral supernatant made in DMEM supplemented with 10% TET-system approved FBS (Clontech #631106) and 1% Penicillin/Streptomycin and then allowed to rest 1 day. Then the G401 cells containing the TET-inducible constructs were selected with 0.25mg/ml G418 and grown in DMEM supplemented with 10% TET-system approved FBS and 1% Penicillin/Streptomycin until selection was complete.

extract_protocol:
For ChIP-seq experiments, chromatin was clarified from sonicated nuclear preps and antibodies were added. For ATAC-seq, nuclei were extracted wtih non-ionic detergent in hypotonic buffer and treated with transposase. For PRO-seq, nuclei were extracted through hypotonic lysis with a low amount of non-ionic detergent.
For ChIP-seq experiments, libraries were made using NEBnext Ultra II DNA library Prep and NEBNext Multiplex Oligos for Illumina, for ATAC-seq libraries were made using transposed DNA made Illumina Nextera DNA kit, NEBNext High-Fidelity 2x PCR Master Mix, and Nextera based primers ordered from IDT, and for PRO-Seq, libraries were made using RNA that was made through a biotin-run on reaction.Biotin-RNA was base hydrolyzed, and extracted. 3'-adaptors were ligated to ends and 5'caps were removed, repaired, and then 5'-adaptors were ligated as well. Purified and intact biotin-RNA containing adaptors was used in a reverase transcriptase reaction to generate cDNA and cDNA was used to amplify full library using NEB High Fidelity Phusion polymerase.

treatment_protocol: Once selection was complete, G401 cells containing TET-inducible constructs would be plated for each experiment with 1ug/ml doxycycline for 24hr to induce expression of proteins

tag: transfection

tag: passage

value: ten-thirty


title: Nascent_repliATAC_rep2


title: PRO-seq SNF5 rep2

description:
nuclear RNA
PROseq-pp_gb_counts.txt

tag: cell line


title: Steady_state_rep3


title: PRO-seq OMOMYC rep2

description:
nuclear RNA
PROseq-pp_gb_counts.txt

value: OMOMYC


title: PRO-seq OMOMYC rep1

description:
nuclear RNA
PROseq-pp_gb_counts.txt


title: PRO-seq OMOMYC rep3

description:
nuclear RNA
PROseq-pp_gb_counts.txt


title: PRO-seq SNF5 rep1

description:
nuclear RNA
PROseq-pp_gb_counts.txt


title: PRO-seq EGFP rep3

description:
nuclear RNA
PROseq-pp_gb_counts.txt

value: EGFP


title: PRO-seq EGFP rep2

description:
nuclear RNA
PROseq-pp_gb_counts.txt


title: ChIP-seq MYC SNF5 rep1

value: MYC


title: ChIP-seq MYC SNF5 rep2


title: ChIP-seq MYC OMOMYC rep2


title: ChIP-seq MYC OMOMYC rep1


title: ChIP-seq MYC EGFP rep2


title: ChIP-seq IgG MYC SNF5 rep2

value: IgG


title: 30m_Mature_repliATAC_rep1

value: EdU-labelled genomic DNA


title: ChIP-seq IgG MYC OMOMYC rep2


title: ChIP-seq MYC EGFP rep1


title: ChIP-seq IgG MYC SNF5 rep1


title: ChIP-seq IgG MYC OMOMYC rep1


title: ChIP-seq IgG MYC EGFP rep2


title: ChIP-seq IgG MYC EGFP rep1


title: ATAC-seq SNF5 rep3


title: ATAC-seq SNF5 rep1


title: ATAC-seq SNF5 rep2


title: ATAC-seq OMOMYC rep3


title: ATAC-seq OMOMYC rep2


title: ATAC-seq OMOMYC rep1


title: 2h_Mature_triptolide_repliATAC_rep2

value: 2 hours Mature


title: ATAC-seq EGFP rep3


title: PRO-seq EGFP rep1

description:
nuclear RNA
PROseq-pp_gb_counts.txt


title: ATAC-seq EGFP rep1


title: ATAC-seq EGFP rep2


title: Hnf4αKO_rep3

status: Public on Apr 08 2019

city: Piscataway

first: Michael

middle: P

last: Verzi

state: NJ

address: 145 Bevier Road

department: Genetics

institute: Rutgers, the State University of New Jersey

description: HNF4_gene_exp_Cuffdiff_percondition.diff

data_processing:
FastQC (version 0.11.3) was used to check the quality of raw sequencing reads (fastq)
Alignment: RNA-seq: TopHat2, version 2.1.0; ATAC-seq and ChIP-seq: Bowtie2, version 2.2.6
RNA-seq: Gene and transcript level computation done with CuffQuant, version 2.2.1
RNA-seq: Normalized abundance measurements generated by CuffNorm, version 2.2.1, calculate FPKM values using quartile normalization
RNA-seq: CuffDiff, version 2.2.1, geometric normalization, per-condition dispersion
ATAC-seq and ChIP-seq: Deeptools bamCoverage (v2.4.2, duplicate reads ignored, RPKM normalized and extended reads) was used to generate bigwig files from bam files.
ATAC-seq and ChIP-seq: Model-based Analysis of ChIP-Seq (MACS 1.4.1) was used for peak calling and to generate bed files from aligned reads. Shiftsize parameter was used for MACS based on the fragment size of Pippin Prep.
Genome_build: mm9/hg19

source_name: Hnf4αKO_duodenum epithelium

extract_protocol:
RNAs were extracted from duodenum epithelia of the indicated genotypes (Wild type, Villin-CreERT2; Hnf4αf/f, Hnf4ɣCrispr/Crispr and Villin-CreERT2; Hnf4αf/f; Hnf4ɣCrispr/Crispr) one day following 2 or 3 consecutive days of tamoxifen injection. Uninjected Villin-CreERT2; Hnf4αf/f mice served as WT control. After flushing the freshly harvested duodenum with cold PBS, the epithelia was dissociated from underlying mesenchyme by incubating with 3 mM EDTA/PBS at 4°C as described previously (Perekatt, 2014). The epithelial cells were washed with PBS, and pelleted to remove excess PBS prior to addition of TRIzol for RNA extraction according to manufacturer’s protocols.
Illumina TruSeq RNA Library Prep kit v2

treatment_protocol: Mice were injected i.p. with tamoxifen (50mg/kg/day)  for 3 consecutive days and were sacrificed 1 day after last injection.

value: duodenum epithelium

tag: genotype

value: Villin-CreERT2; Hnf4alphaf/f

value: tamoxifen 50mg/kg/day


title: Hnf4ɣKO_rep2

source_name: Hnf4ɣKO_duodenum epithelium

treatment_protocol: Mice were injected i.p. with oil (vehicle for tamoxifen) for 3 consecutive days and were sacrificed 1 day after last injection.

value: oil vehicle


title: Hnf4αKO_rep1

treatment_protocol: Mice were injected i.p. with tamoxifen (50mg/kg/day)  for 2 consecutive days and were sacrificed 1 day after last injection.


title: Hnf4αKO_Hnf4ɣChIP_rep2

source_name: Hnf4αKO duodenum epithelium

extract_protocol:
Freshly harvested duodenum was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in 3 mM EDTA in PBS at 4 °C. The tissue was then violently shaken to release the epithelium from underlying muscular tissue. The intestine pieces were discard and the supernatant was collected as whole epithelium fraction. Standard procedures were used for ChIP on mouse duodenum epithelial cells. Briefly, cells were isolated by 3mM EDTA/PBS and then cross-linked in 1% formaldehyde for 10 min at 4°C and then for 35 or 50 min at room temperature. Cells were washed with ice-cold PBS, resuspended with lysis buffer (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris pH 8.0, and protease inhibitor cocktails) and further incubated at room temperature for 10 min. Cell lysates were sonicated using a Diagenode Bioruptor to generate 200 bp to 500 bp fragments, as determined by agarose gel electrophoresis. The supernatant of lysates were diluted 8-fold in binding buffer (20 mM Tris pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktails) and incubated with 6 ug of Hnf4α (Santa Cruz sc-6556 X, lot #B1015) or Hnf4ɣ (Santa Cruz sc-6558 X, lot #F0310) antibody coupled to Dynabeads (Invitrogen) at 4°C overnight. The immunoprecipitates were washed 5 times and rotated at 4°C with RIPA buffer (50 mM HEPES pH 7.6, 1 mM EDTA pH 8.0, 0.7% Na-deoxycholate, 1% NP-40, 0.5M LiCl) and a quick wash with TE buffer (0.1 mM EDTA pH 8.0 and 10 mM Tris pH 8.0). The DNA was recovered by incubating overnight in reverse cross-linking buffer (1% SDS, 0.1 M NaHCO3) at 65 °C. The DNA was purified by QIAquick PCR Purification Kit (QIAGEN) and quantified by using Picogreen (Life Technologies). ChIP enrichment was confirmed with qPCR for putative targets prior to library preparation.
Rubicon Genomics ThruPLEX DNA-seq Kit (Illumina)


title: Hnf4αKO_rep2


title: MV4:11 ChIP-seq WDR5-D9E1l C6 rep3

status: Public on Mar 12 2019

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N3_641_D9E1I_peaks.narrowPeak

data_processing:
RNA-seq reads were aligned to the hg19 genome assembly using STAR after adapter trimming, and quantified by featureCounts.
Differential analysis were performed by DESeq2.
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie2.
Peaks were called by MACS2 with a q-value of 0.01.
Differential binding peaks were identified using DiffBind based on consensus peaks occurring at least two samples.
After adapter trimming and low quality sequence removal by cutadapt, PRO-seq reads longer than 15bp were reversed complemented using FastX tools.
Reverse complements of the trimmed reads were aligned to the human genome hg19 using Bowtie2.
Reads mapped to rRNA loci and reads with mapping quality less than 10 were removed.
Bam files were given to NRSA (http://bioinfo.vanderbilt.edu/NRSA/) to estimate RNA polymerase abundance in proximal-promoter and gene body regions of genes, to calculate pausing index and pausing index alterations, and to detect enhancers and quantify eRNA changes.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq: one peak file per Sample
Supplementary_files_format_and_content: RNA-seq: tab-delimited text file include raw counts for each Sample
Supplementary_files_format_and_content: PRO-seq: tab-delimited text file include raw counts for promoter-proximal and gene body region, and density for gene body

growth_protocol: K562 and MV4:11 cells were cultured at 37ºC with 5% CO2 in Roswell Memorial Park Institute (RPMI) media supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 0.1 mg/ml penicillin/streptomycin (P/S).

extract_protocol:
For ChIP-Seq experiments, chromatin was clarified from sonicated cell lysates by centrifugation and incubated overnight with corresponding antibodies. For RNA-Seq, cells were washed in PBS then lysed in 500 uL Trizol and total RNA was isolated using the Zymo Research Direct-zol RNA MiniPrep kit (Zymo Research  #R2050) with on-column DNase digestion following the manufacturers instructions. For PRO-Seq experiments,  nuclei were extracted through hypotonic lysis with a low amount of non-ionic detergent.
For ChIP-Seq experiments, indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol. For RNA-Seq, library preperation was completed by Genewiz using their standard methods. For PRO-Seq experiments, libraries were made using RNA that was made through a biotin-run on reaction. Biotin-RNA was base hydrolyzed, and extracted. 3'-adaptors were ligated to ends and 5'caps were removed, repaired, and then 5'-adaptors were ligated as well. Purified and intact biotin-RNA containing adaptors was used in a reverase transcriptase reaction to generate cDNA and cDNA was used to amplify full library using NEB High Fidelity Phusion polymerase.

treatment_protocol: For ChIP-Seq experiments, cells were treated with either 0.1% DMSO, 2 µM C6, or 2 µM C6nc for 4 hours. For RNA-Seq experiments, cells were treated with either 0.1% DMSO, 2 µM C6, 2 µM C6nc ot 2 µM Nutlin for 72 hours. For PRO-Seq experiments, cells were treated with either DMSO for 1 hour, or C3 for 15 min, 30 min, or 60 min.

value: 2 µM C6 for 4 hours

value: Cell Signaling WDR5 (D9E1I)


title: Hnf4ɣKO_rep3


title: Hnf4αKO_Hnf4ɣChIP_rep1


title: MV4:11 ChIP-seq WDR5-D9E1l C6 rep2

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N2_641_D9E1I_peaks.narrowPeak


title: Hnf4ɣKO_Hnf4αChIP_rep1

source_name: Hnf4ɣKO duodenum epithelium


title: MV4:11 RNA-seq DMSO/Nutlin Nutlin rep3

description:
Cells were treated for 72 hours. Cells were washed in PBS then lysed in 500 uL Trizol. Total RNA was isolated using the Zymo Research Direct-zol RNA MiniPrep kit with on-column DNase digestion following the manufacturers instructions. Library preparation with rRNA depletion and paired-end 150 base pair sequencing on an Illumina HiSeq 30000 was performed by GENEWIZ. Prior to sequencing RNA integrity was assesed by 2100 Bioanlyser (Agilent) and concentration by Qubit assay.
MV411_DMSO-Nutlin_RNA-seq.count

value: 2 uM Nutlin-3 for 72 hours


title: MV4:11 ChIP-seq WDR5-429A DMSO rep2

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N2_DM_429A_peaks.narrowPeak

value: 0.1% DMSO for 4 hours

value: Bethyl WDR5 (A302-429A)


title: MV4:11 ChIP-seq WDR5-D9E1l C6 rep1

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N1_641_D9E1I_peaks.narrowPeak


title: CaCO2_Avi_Smad4_BiotinChIP

source_name: CaCO2 cells

growth_protocol: Caco-2 cells were cultured in DMEM (Gibco 11995-065), containing 10% FBS (Gibco 26140-095) and 1% penicillin and streptomycin (Invitrogen 15140-122). Cells stably expressing both avi-tagged Smad4 and BirA were selected with media containing 2 ug/ml puromycin and 0.4 mg/ml Hygromycin B, respectively. Cells were fed 24 hours prior to harvest at ~95% confluency.

extract_protocol:
To perform SMAD4 ChIP in Caco-2 cells, each 100 μl pellet were incubated with 1 ml of cross linking solution containing equal amount of DSG (Thermo 20593), DSS (Thermo 21655) and EGS (Thermo 21565) with a final concentration of 2mM NHS ester (reactive groups). Cells were rocked for 20 min at room temperature, after which fresh formaldehyde was added to a concentration of 1.22% formaldehyde, and rocked for an additional 20 min. The cross-linking reaction was stopped with 125 mM glycine, washed with PBS and, nuclei extracted using Downs’ homogenizer and Farnham lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP40, and protease inhibitor cocktails). IP material was subsequently handled as described above for conventional ChIP of sonicated genomic DNA (a final concentration of 0.2% SDS in the sonicates). The diluted sonicates were then incubated with pre-blocked (0.5% BSA/PBS) Streptavidin beads (Invitrogen) overnight at 4°C. The beads were serially washed in low salt (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 150 mM NaCl, 1mM EDTA, and 20 mM HEPES, pH 8.0), high-salt (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 500 mM NaCl, 1mM EDTA, and 20 mM HEPES, pH 8.0), LiCl biuffer (250 mM LiCl, 0.5% Sodium Deoxycholate, 0.5% NP40, 1mM EDTA, and 20 mM HEPES, pH 8.0), and final wash buffer (1mM EDTA, and 20 mM HEPES, pH 8.0), and incubated at 65 °C for 6h in reverse cross linking buffer (0.1mM NaHCO3, 0.01% SDS).
Rubicon Genomics ThruPLEX DNA-seq Kit (Illumina)

value: colon  epithelial cell line (colorectal adenocarcinoma)

value: CaCO2_Avi_Smad4_Biotin cells


title: MV4:11 RNA-seq DMSO/Nutlin Nutlin rep1


title: MV4:11 RNA-seq DMSO/Nutlin DMSO rep3

value: 0.1% DMSO for 72 hours


title: MV4:11 RNA-seq DMSO/Nutlin Nutlin rep2


title: MV4:11 ChIP-seq WDR5-429A DMSO rep1

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N1_DM_429A_peaks.narrowPeak


title: WT_Villi_ATAC

source_name: WT duodenum villi

extract_protocol:
Freshly harvested duodenum was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in 3 mM EDTA in PBS at 4 °C. The tissue was then violently shaken to release the epithelium from underlying muscular tissue. The intestine pieces were discard and the supernatant was collected as whole epithelium fraction. 70-μm cell strainer was used to separate the villi and crypt fractions. Villi were collected from the top of a 70-μm cell strainer, while crypts passed through. Cells were pelleted by centrifugation at 170g at 4 °C, and followed by cold PBS wash. For ATAC-seq, duodenum villi and crypts were treated with pre-warmed 0.25% Trypsin for 8 mins at 37°C on a vortex station, neutralized with 10% FBS, and passed through a 40-μm cell strainer to obtain single cells. 50,000 cells were used for ATAC-seq as described previously with slight modifications. Briefly, cells were centrifuged at 500g for 5mins at 4°C, and resuspended in ice-cold lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% NP-40), followed by centrifugation at 500 g for 10 minutes at 4°C. The isolated nuclear pellets were incubated with a 50 μl reaction of Nextera Tn5 Transposase (Illumina FC-121-1030) for 30 min at 37°C. The transposed chromatin was purified with QIAquick PCR Purification Kit (QIAGEN) and PCR amplified with high-fidelity 2X PCR Master Mix (New England Biolabs M0541). The optimum number of additional cycles was based on 1/3 of the maximum fluorescent intensity. The PCR amplified libraries were purified, fragment size was selected using Pippin Prep, and sequenced on Illumina HiSeq.
The transposed DNA was amplified using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs), and 75 bp single-end reads were sequenced on an Illumina NextSeq 500 instrument.

value: duodenum villi

value: WT


title: MV4:11 RNA-seq DMSO/Nutlin DMSO rep2


title: MV4:11 ChIP-seq WDR5-429A DMSO rep3

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N3_DM_429A_peaks.narrowPeak


title: MV4:11 RNA-seq DMSO/Nutlin DMSO rep1


title: MV4:11 ChIP-seq WDR5-D9E1l C6nc rep3

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N3_566_D9E1I_peaks.narrowPeak

value: 2 µM C6nc for 4 hours


title: MV4:11 RNA-seq C6 rep5

description:
Cells were treated for 72 hours. Cells were washed in PBS then lysed in 500 uL Trizol. Total RNA was isolated using the Zymo Research Direct-zol RNA MiniPrep kit with on-column DNase digestion following the manufacturers instructions. Library preparation with rRNA depletion and paired-end 150 base pair sequencing on an Illumina HiSeq 30000 was performed by GENEWIZ. Prior to sequencing RNA integrity was assesed by 2100 Bioanlyser (Agilent) and concentration by Qubit assay.
K562-MV411_RNA-seq.count

value: 2 uM C6 for 72 hours


title: Hnf4ɣKO_rep1

treatment_protocol: Mice were injected i.p. with oil (vehicle for tamoxifen) for 2 consecutive days and were sacrificed 1 day after last injection.


title: 2h_Mature_DMSO_unbound_rep1

value: 2 hours Mature

value: DMSO control

value: genomic DNA


title: MV4:11 ChIP-seq WDR5-D9E1l C6nc rep1

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N1_566_D9E1I_peaks.narrowPeak


title: MV4:11 ChIP-seq WDR5-D9E1l DMSO rep2

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N2_DM_D9E1I_peaks.narrowPeak


title: MV4:11 RNA-seq C6 rep3


title: MV4:11 RNA-seq C6nc rep5

value: 2 uM C6nc for 72 hours


title: MV4:11 RNA-seq C6 rep4


title: MV4:11 ChIP-seq C6 IgG rep2

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
NA

value: Cell Signaling IgG (2729S)


title: MV4:11 ChIP-seq WDR5-D9E1l C6nc rep2

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N2_566_D9E1I_peaks.narrowPeak


title: MV4:11 RNA-seq C6 rep2


title: MV4:11 RNA-seq C6nc rep3


title: MV4:11 ChIP-seq C6nc IgG rep2


title: MV4:11 ChIP-seq C6 IgG rep1

value: 2 µM C6 for 4 hours


title: MV4:11 ChIP-seq WDR5-D9E1l DMSO rep1

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N1_DM_D9E1I_peaks.narrowPeak

value: Cell Signaling WDR5 (D9E1I)


title: MV4:11 ChIP-seq C6nc IgG rep1


title: MV4:11 ChIP-seq C6nc IgG rep3


title: MV4:11 RNA-seq C6nc rep1


title: MV4:11 RNA-seq C6nc rep4


title: MV4:11 ChIP-seq DMSO IgG rep3


title: MV4:11 RNA-seq C6nc rep2


title: MV4:11 ChIP-seq C6 IgG rep3


title: MV4:11 RNA-seq DMSO rep5


title: MV4:11 RNA-seq DMSO rep4


title: MV4:11 RNA-seq DMSO rep3


title: MV4:11 ChIP-seq WDR5-D9E1l DMSO rep3

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N3_DM_D9E1I_peaks.narrowPeak


title: MV4:11 ChIP-seq DMSO IgG rep2


title: MV4:11 RNA-seq DMSO rep1


title: MV4:11 RNA-seq DMSO rep2


title: K562 RNA-seq C6 rep5


title: K562 RNA-seq C6 rep4


title: K562 RNA-seq C6 rep3


title: K562 RNA-seq C6 rep2


title: K562 RNA-seq C6 rep1


title: K562 RNA-seq C6nc rep5


title: K562 RNA-seq C6nc rep3


title: K562 RNA-seq C6nc rep4


title: K562 RNA-seq DMSO rep3


title: MV4:11 PRO-seq C3-60min rep1

description:
Cells were treated with C3 for 60 min. Cells were washed with PBS and nuclei were isolated. Nuclear runon assay was performed with biotin tagged CTP. Newly synthesized RNA was pulled down using streptavidin beads. Adapters were ligated and library was made. The RNA was then reversce transcriped, amplified and run on gel. 140 to 300 bp smear was extracted from gel and submitted for highthrougput sequencing
PROseq-pp_gb_counts.txt

data_processing:
Library strategy: PRO-Seq
RNA-seq reads were aligned to the hg19 genome assembly using STAR after adapter trimming, and quantified by featureCounts.
Differential analysis were performed by DESeq2.
ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie2.
Peaks were called by MACS2 with a q-value of 0.01.
Differential binding peaks were identified using DiffBind based on consensus peaks occurring at least two samples.
After adapter trimming and low quality sequence removal by cutadapt, PRO-seq reads longer than 15bp were reversed complemented using FastX tools.
Reverse complements of the trimmed reads were aligned to the human genome hg19 using Bowtie2.
Reads mapped to rRNA loci and reads with mapping quality less than 10 were removed.
Bam files were given to NRSA (http://bioinfo.vanderbilt.edu/NRSA/) to estimate RNA polymerase abundance in proximal-promoter and gene body regions of genes, to calculate pausing index and pausing index alterations, and to detect enhancers and quantify eRNA changes.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq: one peak file per Sample
Supplementary_files_format_and_content: RNA-seq: tab-delimited text file include raw counts for each Sample
Supplementary_files_format_and_content: PRO-seq: tab-delimited text file include raw counts for promoter-proximal and gene body region, and density for gene body

molecule: nuclear RNA

value: C3 for 60 min


title: K562 RNA-seq DMSO rep4


title: MV4:11 PRO-seq C3-15min rep2

description:
Cells were treated with C3 for 15 min. Cells were washed with PBS and nuclei were isolated. Nuclear runon assay was performed with biotin tagged CTP. Newly synthesized RNA was pulled down using streptavidin beads. Adapters were ligated and library was made. The RNA was then reversce transcriped, amplified and run on gel. 140 to 300 bp smear was extracted from gel and submitted for highthrougput sequencing
PROseq-pp_gb_counts.txt

value: C3 for 15 min


title: K562 RNA-seq DMSO rep5


title: K562 RNA-seq DMSO rep2


title: K562 RNA-seq DMSO rep1


title: MV4:11 ChIP-seq DMSO IgG rep1

value: 0.1% DMSO for 4 hours


title: MV4:11 PRO-seq C3-15min rep1


title: MV4:11 PRO-seq C3-60min rep2


title: MV4:11 PRO-seq C3-30min rep1

description:
Cells were treated with C3 for 30 min. Cells were washed with PBS and nuclei were isolated. Nuclear runon assay was performed with biotin tagged CTP. Newly synthesized RNA was pulled down using streptavidin beads. Adapters were ligated and library was made. The RNA was then reversce transcriped, amplified and run on gel. 140 to 300 bp smear was extracted from gel and submitted for highthrougput sequencing
PROseq-pp_gb_counts.txt

value: C3 for 30 min


title: MV4:11 PRO-seq C3-00min rep2

description:
Cells were treated with DMSO for 1 hour. Cells were washed with PBS and nuclei were isolated. Nuclear runon assay was performed with biotin tagged CTP. Newly synthesized RNA was pulled down using streptavidin beads. Adapters were ligated and library was made. The RNA was then reversce transcriped, amplified and run on gel. 140 to 300 bp smear was extracted from gel and submitted for highthrougput sequencing
PROseq-pp_gb_counts.txt

value: DMSO for 60 min


title: K562 ChIP-seq WDR5-D9E1l C6 rep1

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N1_641_peaks.narrowPeak


title: K562 ChIP-seq WDR5-D9E1l C6nc rep2

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N2_566_peaks.narrowPeak


title: MV4:11 PRO-seq C3-30min rep2


title: K562 ChIP-seq WDR5-D9E1l C6nc rep1

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N1_566_peaks.narrowPeak


title: K562 ChIP-seq WDR5-D9E1l DMSO rep2

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N2_DM_peaks.narrowPeak


title: K562 RNA-seq C6nc rep2


title: K562 RNA-seq C6nc rep1


title: K562 ChIP-seq C6 IgG rep1


title: K562 ChIP-seq DMSO IgG rep2


title: K562 ChIP-seq DMSO IgG rep1


title: K562 ChIP-seq WDR5-D9E1l C6 rep2

description:
Cells were cross-linked for 10 minutes in 1% formaldehyde and cross-linking quenched with 125 mM glycine. Cells were lysed and chromatin sheared to achieve a mean fragment length of 250 nucleotides. Samples were cleared by centrifugation and lysates incubated overnight with corresponding antibodies. Indexed libraries were made from precipitated DNA using the DNA Ultra II Kit (NEB) per the manufacturer's protocol and libraries assessed using the 2100 Bioanalyzer (Agilent). Libraries were quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and libraries subjected to single-end sequencing according to manufacturer's protocol (Illumina).
N2_641_peaks.narrowPeak


title: MV4:11 PRO-seq C3-00min rep1