GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3176991

Query DataSets for GSM3176991

Status

Public on Mar 12, 2019

Title

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

Sample type

SRA

Source name

MV4:11

Organism

Homo sapiens

Characteristics

cell line: MV4:11
treatment: 0.1% DMSO for 4 hours
chip antibody: Bethyl WDR5 (A302-429A)

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.

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).

Extracted molecule

genomic DNA

Extraction 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.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 3000

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

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

Submission date

Jun 05, 2018

Last update date

Aug 27, 2019

Contact name

Jing Wang

Organization name

Vanderbilt University Medical Center

Street address

2220 Pierce Avenue

City

Nashville