|
| Status |
Public on Jan 17, 2013 |
| Title |
PU.1_stat1ko_ctrl_IFNg_4h |
| Sample type |
SRA |
| |
|
| Source name |
Primary bone marrow-derived macrophages, IFNg for 4hrs, PU.1 ChIP
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: 129S6/SvEv
cell type: bone marrow-derived macrophages (7th day of differentiation)
treatment: IFNg (100 ng/ml) for 4hrs
chip antibody: anti-PU.1 (rabbit polyclonal against the N-terminus (aa. 1-100))
antibody vendor, catalog #: in-house
|
| Treatment protocol |
Macrophages were subjected to different treatments (see individual samples for details).
|
| Growth protocol |
Bone marrow cells isolated from 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 were reversed by incubation overnight at 65ºC. DNA was then purified by QIAquick columns (Qiagen) and quantified using PicoGreen (Invitrogen). ChIP DNA was prepared for Illumina HiSeq 2000 or Genome Analyzer II sequencing using a standard protocol consisting of blunting, addition of dA overhangs, ligation of Illumina adapters, selection on gel and PCR with index primers. A mixture of T4 DNA polymerase, DNA polymerase I and T4 kinase was used according to the manufacturer's instructions. Library preparation was carried out on SPRIworks Fragment Library System.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Chromatin IP against PU.1.
|
| Data processing |
Reads were quality filtered according to the Illumina pipeline.
Reads were mapped to the mouse mm9 genome using Bowtie 0.12.7 (PMID 19261174). All reads with a unique match to the genome with two or fewer mismatches were retained.
Peak calling was performed using MACS 1.4 (PMID 18798982) with default parameters and bw=100. Each IP was compared to input DNA derived from BMDM (GEO Sample GSM487453).
Genome_build: mm9 (NCBI Build 37)
Supplementary_files_format_and_content: *_peaks.bed: List of the peaks called by MACS.
|
| |
|
| Submission date |
Oct 17, 2012 |
| 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 |
GPL13112 |
| Series (2) |
| GSE38377 |
Latent enhancers unveiled by stimulation expand and adapt the available cis-regulatory repertoire (ChIP-seq) |
| GSE38379 |
Latent enhancers unveiled by stimulation expand and adapt the available cis-regulatory repertoire |
|
| Relations |
| SRA |
SRX197252 |
| BioSample |
SAMN01767799 |
