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Sample GSM3177033

Query DataSets for GSM3177033

Status

Public on Mar 12, 2019

Title

MV4:11 PRO-seq C3-30min rep2

Sample type

SRA

Source name

MV4:11

Organism

Homo sapiens

Characteristics

cell line: MV4:11
treatment: C3 for 30 min
chip antibody: N/A

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

nuclear RNA

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

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina NextSeq 500

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

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

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