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Sample GSM385507 Query DataSets for GSM385507
Status Public on Mar 23, 2009
Title HeLa S3 STAT1 ChIP IFNgamma_5
Sample type SRA
 
Source name HeLa S3
Organism Homo sapiens
Characteristics ChIP Antibody: STAT1
Cell Line: HeLa S3
Treatment: IFNgamma stimulated
Extracted molecule genomic DNA
Extraction protocol To prepare immunoprecipated DNA for 1G sequencing, we size-fractionated 5?50 ng of immunoprecipate by 12% PAGE and excised a gel slice containing the 100?300 bp fragments. We eluted DNA from the gel slice overnight at 4 °C in 300 mul of elution buffer (5:1, LoTE buffer (3 mM Tris-HCl (pH 7.5), 0.2 mM EDTA)-7.5 M ammonium acetate) and recovered the DNA using a QIAquick PCR purification kit (Qiagen). Then we repaired the DNA ends using a 1:5 mixture of T4 and Klenow DNA polymerases (Illumina) following the manufacturer's instructions. After a 30-min incubation at 20 °C, we subjected the reaction to phenol?chloroform?isoamyl alcohol (pH 8.0; 100 mul; Fisher) extraction in 0.5-ml phase-lock gel tubes (heavy; Eppendorf) and precipitated the reactions by adding 250 ml of 100% EtOH, 3 ml of mussel glycogen (Invitrogen) and 10 ml of 7.5 M NH4OAc, and incubating at -20 °C for 20 min. We recovered the precipitate by centrifugation at 20,200g for 15 min at 4 °C in an benchtop refrigerated centrifuge (Eppendorf model 5417R). We added a single adenine base to the DNA using Klenow exo? (3' and 5' exo minus; Illumina) following the manufacturer's instructions.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer
 
Description HS0150
Data processing We extracted sequences from the resulting image files using the open source Firecrest and Bustard applications on a 32-CPU cluster running Red Hat Enterprise Linux 4 and Sun Microsystems Grid Engine 6. Reads were aligned (mapped) to the unmasked human reference genome (NCBI v36, hg18) using the Eland application (Illumina). Eland achieves high throughput by permitting no more than two mismatches per read sequence. We maximized the number of mapped reads by iteratively discarding two bases from the end of a rejected read and resubmitting the truncated read to Eland, until either all reads had been aligned to the genome or the lengths of all unmapped reads were less than 20 bp. Only uniquely mapped reads were retained. DNA fragments were represented by a mapped sequence read of length 27 bp. Because a relatively small number of PCR cycles was used in library preparation and DNA-fragment end locations were weakly clustered rather than random (data not shown), we allowed reads with identical start coordinates to be present in a profile. We combined uniquely aligned reads from the three biological replicates into a single set of reads. We transformed mapped sequence reads into profiles of the number of overlapped DNA fragments at each nucleotide in the reference genome. Because a 27-bp read directionally represents one end of a DNA fragment (SET), we approximated the fragment that produced a read sequence by extending the read to generate an XSET. We chose the XSET length to be the mean fragment length of the size selected DNA. From distances between mapped reads, we estimated this length to be 174 bp. A peak's height was the maximum number of overlapped XSETs for that peak. A random expectation for the probability of observing peaks with a particular height was generated from a numerical background model that generated peaks by randomly placing onto a hypothetical genome several fragments equivalent to the actual uniquely mapped number. Each fragment's length was the estimated mean fragment length, that is, the XSET length. Because 27-bp reads can be mapped uniquely to approx90% of the human genome (data not shown), the background simulations used a mappable genome length that was 90% of 3.08 Gb. For a peak height, we estimated the FDR as the ratio of the number of peaks that the background model indicated should occur by chance, to the number observed.
 
Submission date Mar 23, 2009
Last update date May 15, 2019
Contact name Eric Chuah
E-mail(s) echuah@bcgsc.ca
Phone 604-707-5900 3231
Organization name BC Genome Sciences Centre
Street address 570 West 7th Ave
City Vancouver
State/province BC
ZIP/Postal code V5Z 4S6
Country Canada
 
Platform ID GPL9052
Series (1)
GSE15353 Genome-wide profiles of STAT1 DNA association using chromatin immunoprecipitation and massively parallel sequencing
Relations
SRA SRX003321
BioSample SAMN02197853

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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