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Sample GSM929915

Query DataSets for GSM929915

Status

Public on Jul 10, 2012

Title

Input_DNA

Sample type

SRA

Source name

UVSS1KO Cells

Organism

Homo sapiens

Characteristics

cell type: SV40 Immortalized Fibroblast
chip antibody: none

Growth protocol

UVSS1KO cells expressing CSB-PGBD3 were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS, penicillin, and streptomycin with 200 µg/mL hygromycin.

Extracted molecule

genomic DNA

Extraction protocol

Nuclei crosslinked with 0.5% formaldehyde were sonicated and precleared with crosslinked Staph A cells. ChIPs were performed with a 1:200 dilution of mouse monoclonal 1B1 directed against the N-terminus of CSB (Hua-Ying Fan, University of Pennsylvania) precipitated using Protein G Dynabeads (Invitrogen). The input sample was digested with RNase, protease, and decrosslinked without enrichment by ChIP. The input sample was digested with RNase, protease, and decrosslinked without enrichment by ChIP. The ends of the ChIP and input samples were repaired using End-It (Epicentre), A-tailed using Taq polymerase (Invitrogen), and Illumina paired-end sequencing adapters were ligated using Quick T4 DNA Ligase (NEB). DNA fragments ranging from 400 to 700 bp were selected and purified by PAGE, then preamplified for 9 (input) or 12 cycles (ChIP) using Illumina paired-end preamplification primers and BioRad iTaq supermix. The preamplified samples were purified using a Qiagen PCR Cleanup kit and sequenced on the Genome Analyzer.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer II

Data processing

Base calls were made using Illumina RTA 1.5
Raw reads were aligned to the hg18 human genome using Bowtie v0.12.7 with settings -n 0 -m 1 --chumkmbs=256 -S to SAM format
A fragment map was generated using a PERL script that treated paired end reads as the boundaries of IPed fragments. Only regions of at least 5 overlapping fragments were mapped to a WIG file.
Peaks were called using MACS v1.4 with a p-value < 1e-12, ERANGE v3.2.1 with the setting -minimum 2 and QuEST v2.4 with settings for transcription factor ChIP and custom peak calling parameters (20, 10, 3)
Summits were located for peaks identified by all 3 peak callers using a PERL script that searched for the highest point of fragment overlap in each common peak
Genome_build: hg18
Supplementary_files_format_and_content: WIG file was generated using a PERL script that treated paired end reads as the boundaries of IPed fragments. Only regions of at least 5 overlapping fragments were mapped. Scores represent the number of fragments overlapping each genomic position.

Submission date

May 11, 2012

Last update date

May 15, 2019

Contact name

Lucas T Gray

E-mail(s)

lucasg@alleninstitute.org

Organization name

Allen Institute for Brain Science

Department

Molecular Genetics