|
| Status |
Public on Nov 10, 2014 |
| Title |
BRD4_serum_DMSO |
| Sample type |
SRA |
| |
|
| Source name |
NIH3T3 fibroblasts
|
| Organism |
Mus musculus |
| Characteristics |
culture condition: serum starved and re-fed
chemicals: DMSO
chip antibody: BRD4 (C-terminus) made in house
|
| Treatment protocol |
1. NIH3T3 cells were serum-starved in the presence of 0.5% serum for 48 hr, preincubated with JQ1 (500 nM) or vehicle (DMSO) for 30 min; and stimulated with 15% serum for 30 min or left unstimulated in the continued presence of JQ1 or vehicle. Control cells were cultured in the continuous presence of 10% serum.
2. NIH3T3 cells were sequentially infected with shRNA retrovirus (pSRneo-shBRD4) and YFP or YFP-BRD4 expressing retrovirus (pMSCVpuro-YFP, pMSCVpuro-YFP-BRD4, pMSCVpuro-YFP-BRD4short or pMSCVpuro-YFP-BRD4short-mBD) in the presence of polybrene. The transduced cells were selected with G418 and puromycin.
|
| Growth protocol |
NIH3T3 cells were serum-starved in the presence of 0.5% serum for 48 hr, preincubated with JQ1 (500 nM) or vehicle (DMSO) for 30 min; and stimulated with 15% serum for 30 min or left unstimulated in the continued presence of JQ1 or vehicle. Control cells and cells expressing YFP-tagged constructs were cultured in the continuous presence of 10% serum.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq: Cells were chemically cross linked by 1% formaldehyde. Cell lysates were made by appropriate sonication and protein-DNA complexes were isolated with antibody.
ChIP-seq: Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired and the blunt, phosphorylated ends were treated with Taq polymerase 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 ~250 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 HiSeq 2000 or 2500 following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
exp_set#59-2_CHIP
|
| Data processing |
alignment, ChIP-seq: Sequence reads were obtained and mapped to the mouse mm9 genomes using the Illumina Pipeline. All reads mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 200 bp (SICER).
peaks, ChIP-seq: Peak detection was performed with SICER (Zang et al, 2009 Bioinformatics, 25:1952-1958).
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph for peak files, bigWig for viewing strand specific nuclear RNA data with the UCSC genome browser
|
| |
|
| Submission date |
Jun 23, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Yuka Kanno |
| E-mail(s) |
kannoy@mail.nih.gov
|
| Phone |
301-402-3008
|
| Organization name |
NIH
|
| Department |
NIAMS
|
| Lab |
LCBS-MIIB
|
| Street address |
10 Center Drive, Rm13C120
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892-1930 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE58731 |
BRD4 assists elongation of both coding and enhancer RNAs guided by histone acetylation |
|
| Relations |
| BioSample |
SAMN02869893 |
| SRA |
SRX620283 |
