|
| Status |
Public on Mar 22, 2010 |
| Title |
GM18526_NFkB_II_ChIPSeq_IgG_Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Lymphoblastoid Cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: GM18526
cell type: Lymphoblastoid cell
treatment: TNF-alpha induced (6h)
chip antibody: normal rabbit IgG
|
| Biomaterial provider |
Coriell Cell Repositories http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM18526
|
| Treatment protocol |
Prior to cross-linking, cells were treated with 25 ng/mL human recombinant TNF-alpha (eBioscience #14-8329) for six hours at 37°C, 5% CO2.
|
| Growth protocol |
Lymphoblastoid cell lines were grown in RPMI-1640 Medium, supplemented with glutamine, 15% FBS, and antibiotics (penicillin-streptomycin) at 37C in 5% CO2, to a density of 6-8 x10^5 cells/ml.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chomatin immunoprecipitation was performed as previously described (Euskirchen et al. 2007; Rozowsky et al. 2009). 2 x 10^8 cells were cross-linked in 1% formaldehyde for 10 minutes at room temperature. The cross-linking reaction was quenched by adding glycine to a final concentration of 125 mM. Nuclear lysates were sonicated using a Branson 250 Sonifier (power setting 2, 100% duty cycle for 7 × 30-s intervals), such that the chromatin fragments ranged from 50-2000kb. Clarified lysates were divided in half and treated overnight at four degrees Celsius with 8μg of either anti-NFκB p65 (C-20) rabbit polyclonal antibody (Santa Cruz sc-372) or normal rabbit IgG (Santa Cruz Sc-2027). Protein-DNA complexes were captured on Protein A agarose beads (Upstate #16-156) and eluted in 1% SDS TE buffer at 65°C. Following cross-link reversal and purification, the ChIP DNA sequencing libraries were generated according to Illumina DNA Sample Kit Instructions (Illumina Part # 0801– 0303). The protocol was modified such that enzymes were obtained from other suppliers, as described in Auerbach et al. 2009.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against normal rabbit IgG
|
| Data processing |
FASTQ files are either the sequence.txt files from the Illumnia pipeline (which exclude low quality reads) or, when available, are generated from the Illumina export.txt files which include all reads. Both types of alignment files, eland_result.txt and eland_multi.txt, are directly out of the Illumnia pipeline and unmodified. The narrowPeak files are the scored results generated from the PeakSeq pipeline (http://0-www-ncbi-nlm-nih-gov.brum.beds.ac.uk/pubmed/19122651?dopt=Abstract) and formatted in the UCSC narrowPeak format.
|
| |
|
| Submission date |
Dec 15, 2009 |
| 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) |
| GSE19485 |
Human variation in PolII and NF-KappaB binding (ChIP-seq study with NF-KappaB) |
| GSE19486 |
Human variation in PolII and NF-KappaB binding |
|
| Relations |
| SRA |
SRX017903 |
| BioSample |
SAMN00010278 |
