|
| Status |
Public on Nov 24, 2010 |
| Title |
macrophage_Pol_II_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
primary bone marrow-derived macrophages
|
| Organism |
Mus musculus |
| Characteristics |
cell type: primary bone marrow-derived macrophages
strain: C57BL/6
treatment: unstimulated macrophages
donor age: 10 month old male
chip antibody: Pol_II
|
| Treatment protocol |
Cells were fixed with 2 mM disuccinimidyl glutarate for 30 minutes, then with 1% formaldehyde for 10 minutes, then glycine-quenched and harvested.
|
| Growth protocol |
Bone marrow was purified and differentiated in DMEM containing 20% fetal bovine serum, 30% L929 conditioned media, and antibiotics for 5 days, then re-plated in macrophage serum free media (Invitrogen) overnight.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the ChIP seq sample prep kit (IP-102-1001). 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 200-400 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 |
Chromatin IP against Pol II
processed data file (alignment): s_2_export.txt
processed data file (peaks): s_2_peaks.txt
raw data file: s_2_traces.srf
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the mouse genome (July 2007; mm9, NCBI Build 37) using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 200 bp to create summary windows.
Peaks: Peak detection was performed with the HOMER software suite (http://biowhat.ucsd.edu/homer/).
|
| |
|
| Submission date |
Jun 19, 2009 |
| Last update date |
May 15, 2019 |
| Contact name |
Ruth T Yu |
| Organization name |
Salk Institute
|
| Department |
Gene Expression Lab
|
| Lab |
Ronald Evans
|
| Street address |
10010 N Torrey Pines Rd
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE16723 |
Bcl6 and NFkB cistromes mediate opposing reulation of the innate immune response |
|
| Relations |
| SRA |
SRX016347 |
| BioSample |
SAMN00008433 |
