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Sample GSM1598304

Query DataSets for GSM1598304

Status

Public on Feb 03, 2015

Title

SW480_Input pooled reps

Sample type

SRA

Source name

Human colorectal cancer cell line

Organism

Homo sapiens

Characteristics

cell line: SW480
antibody: none

Growth protocol

Plated to mid-confluency, medium change every 3-5 days. 5 x 175mm3 flasks harvested for each experiment.

Extracted molecule

genomic DNA

Extraction protocol

For chromatin: Mnase treatment followed by mild crosslinking and low salt extraction overnight at 4C. IPs with CENP-A, HJURP or Mock. For DHS fragments: Low DNAse I treatment followed by sucrose gradient purification of DHS fragments. Please see Athwal-Walkiewicz et al Epigenetics and Chromatin 8:3 for full protocol and cited references.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP 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 for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer IIx

Data processing

Bioinformatic analysis of chromatin immunoprecipitation-seq, DNase-seq, and TOMTOM DNA motif enrichment :Purified DNA from ChIP or DNase I digested chromatin were used to prepare libraries for Illumina high-throughput sequencing as described in manufacturer’s protocol (Illumina Sequencing, San Diego, CA, USA). Libraries were sequenced to generate 35 bp single end reads using Illumina GAII sequencer at the Advanced Technology Center, NCI (Frederick, MD). Sequence reads were mapped to the reference genome hg19 by the CASAVA 1.8.2 pipeline.
Hotspot detection for DNase-seq : Regions of local enrichment of sequence tags using a hotspot detection algorithm was identified essentially as previously described [55,77] with a false discovery rate (FDR) of 0.1%.
Hotspot detection and input adjustment for chromatin immunoprecipitation-seq: The hotspot detection algorithm was similarly applied to ChIP-seq data with the following modification. The sequencing data from matching input samples are used for the processing of ChIP data, as a measure of background signal that might be significant. After normalizing the input data to match the number of tags in the ChIP data, the number of input tags is subtracted from the number of ChIP tags in the target window before calculating its z-score.
Genome_build: GRCh37/hg19
Supplementary_files_format_and_content: CSV (Comma-Separated Values)

Submission date

Jan 30, 2015

Last update date

May 15, 2019

Contact name

Songjoon Baek

Organization name

NCI / NIH

Department

CCR

Lab

LRBGE