|
| Status |
Public on Mar 30, 2020 |
| Title |
ChIP-Seq_MCF7_Med12_siCTL |
| Sample type |
SRA |
| |
|
| Source name |
breast cancer
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
chip antibody: A399-774A
treatment: ethanol+siCTL
|
| Treatment protocol |
MCF7 cells were maintained in stripping medium (phenol red free) for three days before treating with or without estrogen (E2, 10-7 M) for 1h.
|
| Growth protocol |
MCF7 cells were cultured in DMEM medium supplemented with 10% FBS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP assays, cells were maintained in stripping medium (phenol red free) for three days before treating with or without estrogen (E2, 10-7 M) for 1 hr. Cells were then fixed with 1% formaldehyde (Sigma) for 10 mins at room temperature (RT) (for MED12 ChIP), or fixed with disuccinimidyl glutarate (DSG) (2 mM) (Proteochem) for 45 mins at RT, washed twice with PBS and then double-fixed with 1% formaldehyde for another 10 mins at RT (for CARM1 ChIP). Fixation was stopped by adding glycine (0.125 M) and incubated for 5 mins at RT, followed by washing with PBS twice. Chromatin DNA was sheared to 300~500 bp average in size through sonication. Resultant was immunoprecipitated with anti-MED12, anti-Pol II or anti-CARM1 antibody overnight at 4 ℃, followed by incubation with protein G magnetic beads (Bio-Rad, 161-4023) for an additional 2 hrs. After washing and elution, the protein-DNA complex was reversed by heating at 65 ℃ overnight. Immunoprecipitated DNA was purified by using QIAquick spin columns (Qiagen) and subjected to high throughput sequencing.
The libraries were constructed following NEB’s Chip-Seq Sample prep kit. Briefly, Chip DNA was end-blunted and added with an ‘A’ base so the adaptors from Illumina with a ‘T’ can ligate on the ends. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
Basecalling was performed using CASAVA version 1.4
Reads were aligned to the hg19 genome assembly using Bowtie version 2.2.1 with the default options.
The bigwig files were generated by using HOMER, in which the total tags are normalized to 1.00e+07.
Peaks are called by using Homer findPeaks script
Genome_build: hg19
|
| |
|
| Submission date |
Dec 27, 2018 |
| Last update date |
Mar 30, 2020 |
| Contact name |
Jiancheng Ding |
| E-mail(s) |
djch@xmu.edu.cn
|
| Organization name |
Xiamen University
|
| Street address |
School of Pharmaceutical Sciences, Xiamen University Xiang`an South Road
|
| City |
Xiamen |
| State/province |
Fujian |
| ZIP/Postal code |
361102 |
| Country |
China |
| |
|
| Platform ID |
GPL21290 |
| Series (2) |
| GSE124448 |
A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor a-dependent transcriptional activation and breast carcinogenesis (ChIP-Seq) |
| GSE124449 |
A hypermethylation strategy utilized by enhancer-bound CARM1 to promote estrogen receptor a-dependent transcriptional activation and breast carcinogenesis |
|
| Relations |
| BioSample |
SAMN10658631 |
| SRA |
SRX5185602 |
