GSM1619773: Input, untreated, replicate 1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Input control
Antigen
Input control
Cell type
Cell type Class
Breast
Cell type
MDA-MB-231
Primary Tissue
Breast
Site of Extraction
Effusion, Pleural
Tissue Diagnosis
Adenocarcinoma
Attributes by original data submitter
Sample
source_name
MDA-MB-231
cell line
MDA-MB-231
cell type
triple negative breast adenocarcinoma
chip antibody
none (input)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
MDA-MB-231 cells in culture were crosslinked using 1% formaldehyde for 10 minutes at room temperature, following which excess reagent was quenched by the addition of 125 mM glycine for 5 minutes. Cells were washed with ice cold PBS and the pellet was resuspended in ChIP lysis buffer (1% SDS, 10 mM EDTA pH 8.0, 50 mM Tris-HCl pH 8.1, protease inhibitor cocktail and PMSF). DNA was sheared to 200-500bp using a Branson probe sonicator while keeping the sample on ice. A small aliquot of sheared chromatin was saved as “input”. Chromatin from 2 million cells was used for each ChIP reaction and diluted 10-fold using ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, protease inhibitor cocktail and PMSF). Immunoprecipitation was performed by incubating diluted chromatin with Dynabeads G (Invitrogen, pre-washed with BSA/PBS) and the following antibodies overnight at 4°C: H3K9me2 (Abcam, ab1220; 2 mg per ChIP) or H3K9ac (Millipore #07-352; 3.5 ml per ChIP). Beads were subsequently washed using the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), LiCl buffer (0.25M LiCl, 1% IGEPAL-CA630, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and twice with TE (1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Dissociation of chromatin-antibody complexes from the beads was facilitated by eluting twice using an appropriate buffer (1% SDS, 0.1M NaHCO3) for 15 minutes each at RT. Crosslinks in all ChIP and input samples were reversed by incubating with NaCl overnight at 65°C. Samples were treated with RNAse A for one hour at 37°C followed by addition of Proteinase K for one hour at 45°C for cleanup. In the final step, DNA was purified using the MinElute PCR Purification Kit (Qiagen). Libraries were made using the Diagenode MicroPlex Library PreparationTM kit as per manufacturer's protocols.