|
| Status |
Public on May 21, 2010 |
| Title |
polII_ser5.UT |
| Sample type |
SRA |
| |
|
| Source name |
Primary bone marrow-derived macrophages (BMDM) from Fvb mice, untreated, ChIP
|
| Organism |
Mus musculus |
| Characteristics |
strain: Fvb
gender: female
cell type: BMDM cells (7th day of differentiation)
antibody: RNA polymerase II CTD repeat YSPTSPS (phospho S5) – Abcam (ab5131)
treatment: none
|
| Treatment protocol |
Macrophages were stimulated with lipopolysaccharide (LPS, 10 ng/ml).
|
| Growth protocol |
Bone marrow cells isolated from female Fvb mice were plated in 10 cm plates in 5ml of BM-medium (high glucose DMEM supplemented with 20% low endotoxin fetal bovine serum, 30% L929-conditioned medium, 1% glutamine, 1%, Pen/Strep, 0.5% Sodium Pyruvate, 0.1% β-mercaptoethanol). Cultures were fed with 2.5 ml of fresh medium every two days.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP lysates were generated from 2x10^8 cells. Cells were fixed in 1% formaldehyde for 10min. Lysate was immunoprecipitated with 10ug of antibody. Antibodies were pre-bound overnight to 100ul of G protein-coupled paramagnetic beads in PBS/BSA 0.5%. Beads were then added to lysates (the preclearing step was omitted) and incubation was let to proceed overnight. Beads were washed 6 times in a modified RIPA buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and once in TE containing 50mM NaCl. DNA was eluted in TE containing 2% SDS and crosslinks reversed by incubation overnight at 65ºC. DNA was then purified by Qiaquick columns (Qiagen) and quantified using PicoGreen (Invitrogen). ChIP DNA was prepared for Solexa 2G sequencing using a standard protocol consisting of blunting, addition of dA overhangs, ligation of Illumina adapters, selection on gel and PCR. A mixture of T4 DNA polymerase, DNA polymerase I and T4 kinase was used according to manufacturer’s instruction.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against polII ser5.
|
| Data processing |
Alignment: Sequence reads were mapped to the mouse mm9 genome using Bowtie (PMID: 19261174, Illumina pipeline version 1.3). All reads that map uniquely to the genome with two or fewer mismatches were kept. 76-bp reads were trimmed at the 3' end using different cutoffs in order to obtain the highest number of mappable reads onto the genome. In this way, the first 30bp at the 5' end for the untreated sample and the first 45bp at the 5' end for the LPS treated sample were kept.
Peaks: Analyses were performed using MACS (PMID: 18798982) with a p-value threshold of 10^-5. Both ChIPs were normalized against input DNA (GSM487453).
|
| |
|
| Submission date |
Jan 21, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Iros Barozzi |
| E-mail(s) |
iros.barozzi@meduniwien.ac.at
|
| Organization name |
Medical University Vienna
|
| Street address |
Borschkegasse 8a
|
| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
| |
|
| Platform ID |
GPL9250 |
| Series (1) |
| GSE19991 |
A large fraction of extragenic RNA Pol II transcription sites overlap enhancers (2) |
|
| Relations |
| SRA |
SRX017734 |
| BioSample |
SAMN00009907 |
