|
| Status |
Public on Sep 16, 2008 |
| Title |
IFN_gamma_Input_DNA in HeLa S3 |
| Sample type |
SRA |
| |
|
| Source name |
IFN Gamma stimulated HeLa S3 Input DNA
|
| Organism |
Homo sapiens |
| Characteristics |
HeLa S3 cells (ATCC/Snyder Lab)
growth properties: Suspension Morphology: epithelial Treatment: IFN gamma
|
| Treatment protocol |
For STAT1 cells were then stimulated at these same growth conditions with 1000 U/ml (final) of Interferon alpha [PBL Biomedical Laboratories Product #11100-1, Recombinant human IFN alpha (Hu-IFN-alpha-A; Hu-IFN-alpha-2a)].
|
| Growth protocol |
HeLaS3 cells were grown in Minimum Essential Medium modified for Suspension (SMEM, Invitrogen), supplemented with glutamine, 10% FBS, and antibiotics (penicillin-streptomycin) at 37C in 5% CO2, to a density of 5 x10^5 cells/ml.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
STAT1 ChIP DNA samples were prepared from HeLa S3 cells following stimulation with IFN gamma (R&D systems #285-IF) for 30 min as previously described (Hartman et al. 2005; Euskirchen et al. 2007). Lysates were clarified from sonicated nuclei and STAT1-DNA complexes were isolated with anti-STAT1 alpha p91 (C-24) rabbit polyclonal antibody (sc-345 from Santa Cruz Biotechnology). The corresponding Input DNA samples were prepared from the same clarified lysates of the gamma interferon-stimulated, formaldehyde-crosslinked, sonicated HeLa S3 nuclei as were used for the STAT1 ChIP samples. 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 II |
| |
|
| Description |
IFN Gamma stimulated HeLa S3 Input DNA
Treatment: Stimulated with IFN gamma
|
| Data processing |
Enriched peak regions corresponding to transcription factor binding sites were identified by first extending all the uniquely mapped tags reads (mapped against NCBI36) to the averaged sequenced DNA fragment size ~200bps and then accumulated into fragment density signal maps. Peaks in these maps are identified by comparison to a computer simulated null model for each Mb of each chromosome (accounting for the variability in mappability of sequence tags across the human genome. For potential target regions the number of sequenced tags is compared against the normalized number of sequence tags overlapping the region from the matching Input DNA reference sample. Fold enrichment ratios are computed from the ratio of these two sets of counts for each regions as well as p-value using the cumulative distribution function for the binomial distribution. P-value are corrected for multiple hypothesis testing using a Benjamini-Hochberg correction factor and a false discovery rate threshold of 0.05 is imposed. (See Rozowsky et al. submitted)
|
| |
|
| Submission date |
Sep 15, 2008 |
| Last update date |
May 15, 2019 |
| Contact name |
Flora Vaccarino |
| Organization name |
Yale University
|
| Department |
Child Study Center
|
| Lab |
Vaccarino
|
| Street address |
230 South Frontage Road
|
| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06520 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE12782 |
STAT1 transcription factor in Human HeLa S3 |
| GSE12783 |
Genome-wide maps of transcription factor binding using ChIP-Seq for Human ENCODE |
|
| Relations |
| SRA |
SRX003800 |
| BioSample |
SAMN02195760 |
