|
| Status |
Public on Jan 17, 2013 |
| Title |
PU.1_IL1b_4h |
| Sample type |
SRA |
| |
|
| Source name |
Primary bone marrow-derived macrophages (BMDM)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: BMDM cells (7th day of differentiation)
antibody: Anti-PU.1 (in-house rabbit polyclonal against the N-Terminal (aa. 1-100))
treatment: IL1b (10 ng/ml) for 4hrs
|
| Treatment protocol |
Macrophages were subjected to different treatment (see individual samples for details)
|
| Growth protocol |
Bone marrow cells isolated from C57BL/6 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 HiSeq 2000 Illumina sequencing using a standard protocol consisting in 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 manufacturer’s instruction. Library preparation is carried out on SPRIworks Fragment Library System.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against PU.1
|
| Data processing |
Reads 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 (except for the pol_II for which a bw=300 was used). Each IP was compared to input DNA derived from BMDM (GSM487453).
Genome_build: mm9 (NCBI Build 37)
Supplementary_files_format_and_content: *_peaks.bed, list of the peaks called by MACS
|
| |
|
| Submission date |
May 31, 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 |
GPL9250 |
| 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 |
SRX150881 |
| BioSample |
SAMN01001313 |
