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Sample GSM2058108

Query DataSets for GSM2058108

Status

Public on Feb 08, 2017

Title

V389 input DNA

Sample type

SRA

Source name

CRC cell line

Organism

Homo sapiens

Characteristics

tissue: colorectal cancer cell line
cell line: V389

Treatment protocol

For RNA-Seq samples, CRC cells at ~0.5×10^6 cells/well in 6-well plates were treated with 500nM JQ1 for 0.5, 1, 6, or 24 hours, or 0 hours as a control

Growth protocol

CRC cell lines were grown in MEM media supplemented as previously described, except for COLO205 cells which were grown in RPMI-1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin/streptomycin.

Extracted molecule

genomic DNA

Extraction protocol

For ChIP-Seq, lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. For DNase Hypersensitivity, nuclei were isolated and treated with DNase I. For RNA-Seq samples, cells were lysed and RNA extracted with TRIzol according to the manufacturer’s protocol.
ChIP-Seq libraries were prepared with NEB reagents as previously described. Briefly, DNA was end-repaired and the blunt, phosphorylated ends were treated to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers and library fragments of ~250 bp were band isolated from an agarose gel. DNase Hypersensitivity libraries were prepared as previously described. Briefly, DNase I treated DNA was blunt-ended. DNA fragments were ligated to biotinylated linkers and digested with MmeI, producing 2 bp overhangs for ligation with a second linker or Illumina adapters. DNA was then PCR amplified and library fragments of ~86 bp were band isolated from a gel. RNA libraries were prepared for sequencing using standard Illumina protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiScanSQ

Data processing

Adapter and quality trimming of both ChIP-Seq and RNA-Seq reads was done using FASTX Toolkit 0.0.13. Reads below 25bp in length or reads with Phred quality scores below 20 were discarded
ChIP-seq reads were aligned to hg19 with Bowtie2 version 2.0.6, using default parameters. RNA-Seq reads were aligned to hg19 using TopHat v1.3.2
Potential PCR duplicates were removed from aligned ChIP Seq reads files
ChIP-Seq peaks were called using MACS v1.4. Cufflinks v1.3.0, run with genomic bias correction, was used to calculate transcript abundancies in the RNA-Seq data.
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-Seq WIG tracks were generated with MACS v.14. The WIG signal was divided by mean WIG signal for each sample, and the WIGs were converted to bigWIGs

Submission date

Feb 09, 2016

Last update date

May 15, 2019

Contact name

Peter Scacheri

E-mail(s)

pxs183@case.edu

Phone

216-368-3458

Organization name

Case Western Reserve University