|
| Status |
Public on Sep 29, 2014 |
| Title |
CBP_null2_EtOH |
| Sample type |
SRA |
| |
|
| Source name |
primary CBP null MEF (e14.5)
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 x 129Sv (F1)
treatment: 2hrs ethanol vehicle
passage: 5
chip antibody: CBP (CREBBP) Brindle 8690
|
| Treatment protocol |
25ul of 100mM 2',2'-dipyridyl or ethanol vehicle was added to each plate and plates were returned to normoxia 5% CO2 incubator for 2hrs. Medium was then removed from the plates and 15ml 3% paraformaldehyde in PBS was added and cells were allowed to fix for 20min at room temperature. Fixed adherent cells were washed 2x with PBS and scraped from plates into PBS to harvest. Cells were lysed in buffer containing 1% SDS.
|
| Growth protocol |
6 15 cm dishes of each p5 wild type and CBP conditional (flox//flox) MEF were plated at 4x10^6 cells/dish in 25ml DMEM+10%FBS containing Cre recombinase expressing adenovirus at an MOI of 100 and grown in a 3% O2 5%CO2 incubator. Medium was changed 16hrs later for 25ml DMEM + 10%FBS and MEFs were grown for 48hrs in 3% O2 5%CO2 incubator. MEFs were transferred to a normoxia 5% CO2 incubator and grown for 24hrs.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell lysates were sonicated (5x 10sec @ 15micron amplitude, MSE Soniprep 150) and immunoprecipitated using CBP antibody (MEFs lacking CBP provide control for antibody specificity)
library prepared according to Illumina protocol "Preparing Samples for ChIP Sequencing of DNA" (2007). 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 ~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.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.8
ChIP-seq reads were aligned to the mm9 genome assembly using BWA version 0.5.9 with default setting
Duplicate reads as well as reads with map quality score of less than 1 were removed
Peaks were called using MACS (version 1.4.2) with p-value cutoff of 1E-5
Genome_build: mm9
Supplementary_files_format_and_content: bigWig file were generated based on read coverage
|
| |
|
| Submission date |
Jan 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Suzanne J Baker |
| E-mail(s) |
suzanne.baker@stjude.org
|
| Organization name |
St Jude Children's Research Hospital
|
| Department |
Developmental Neurobiology
|
| Street address |
262 Danny Thomas Place
|
| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38105 |
| Country |
USA |
| |
|
| Platform ID |
GPL11002 |
| Series (2) |
| GSE54453 |
CREBBP (CBP) ChIP-seq in wild type and CBP null MEFs +/- dipyridyl |
| GSE54454 |
Genome-wide and single-cell analyses reveal target gene context-dependent relationship between Crebbp recruitment and both constitutive and signal-responsive gene expression. |
|
| Relations |
| BioSample |
SAMN02598602 |
| SRA |
SRX451039 |
