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Sample GSM3593644

Query DataSets for GSM3593644

Status

Public on Nov 08, 2019

Title

RNA-seq HA-WBM-1D2 rep3

Sample type

SRA

Source name

RNA-seq HA-WBM-1D2

Organism

Homo sapiens

Characteristics

cell line: Ramos
cell type: Burkitt's lymphoma cell line
genotype/variation: Tumor derived WBM-HA
treatment: 48h post 4-OHT treatment

Treatment protocol

Cells were plated at 0.75 million in RPMI +10% FBS + Pen/Strep and treated with 20 nM 4-OHT for 24 h

Growth protocol

Ramos cells were maintained in RPMI1640 +10 % FBS, Pen/Strep, and 50 ug/ml Hygromycin B. Puromycin was added to 200 ng/ml for WT, WBM, and ∆264 cells.

Extracted molecule

total RNA

Extraction protocol

Chromatin was clarified from sonicated cells and protein-DNA complexes were isolated with antibody.
ChIP libraries were prepared according to New England Biolabs instructions accompanying the NEBNext Ultar II DNA Library Prep Kit for Illumina (Part# E7645S). After adapter ligation DNA was PCR amplified with NEBNext Multiplex Oligos for Illumina (Part# E7335S and E7500S) for 16 cycles (HA ChIP) or 15 cycles (WDR5 ChIP). For RNA-seq, libraries were prepared using Poly-A selected at Genewiz using their standard methods, 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.RNA libraries were prepared using Poly-A selected RNA.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

processed data file: Lance-RAMOS.count
WBM-1D2-3

Data processing

ChIP-seq reads were aligned to the hg19 genome assembly using Bowtie2.
Reads with mapping quality less than 10 were removed.
Peaks were called by MACS2.
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.
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.
Genome_build: hg19
Supplementary_files_format_and_content:
ChIP-seq: one peak file per Sample
PRO-seq: text file with raw counts for promoter-proximal and gene body region, and density for gene body (ppc, gbc, gbd)
RNA-seq: tab-delimited text file include raw counts for each Sample

Submission date

Feb 07, 2019

Last update date

Nov 08, 2019

Contact name

Jing Wang

Organization name

Vanderbilt University Medical Center

Street address

2220 Pierce Avenue

City

Nashville