GSM3533472: ChIP-Seq MCF7 Med12 siCARM1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
MED12
Cell type
Cell type Class
Breast
Cell type
MCF-7
Primary Tissue
Breast
Site of Extraction
Pleura
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
breast cancer
cell line
MCF7
chip antibody
A399-774A
treatment
ethanol+siRNA
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_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.