GSM4695916: H1299-wtSMARCA4 Dox BRG1 Rep2; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
SMARCA4
Cell type
Cell type Class
Lung
Cell type
NCI-H1299
Primary Tissue
Lung
Tissue Diagnosis
Carcinoma Non-Small Cell
Attributes by original data submitter
Sample
source_name
Lung
biomaterial_provider
ATCC
antibody
BRG1
cell line
NCI-H1299
tissue
Lung
Sex
male
age
43 years adult
source
Non-Small cell lung cancer
treatment
doxycycline
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For ChIP, cells were grown in P-150 cm cell dishes and fixed with 1% methanol-free formaldehyde (Thermo Scientific) for 10 min at room temperature, then quenched by 125 mmol/L glycine for 15 min at room temperature, washed with ice-cold PBS twice and centrifuged at 200g, 4°C for 5 min. The pellet was resuspended in 1 mL of cell lysis buffer (10 mmol/L Tris-HCl, 10 mmol/L NaCl, 0.5% NP-40, protease inhibitor) and kept at 4°C, rotating for 30 min. After centrifugation, the pellet was resuspended in 1 mL of nuclear lysis buffer (1% SDS, 10 mmol/L EDTA, 50 mmol/L Tris-HCl pH 8.0, protease inhibitor) and kept at 4°C for 60 min. After another centrifugation, the lysate was sonicated with a Covaris M220 instrument to yield chromatin fragments of an average size of 0.25–1.00 kb, and then frozen at -20ºC for 30 min. The chromatin was thawed on ice and centrifuged at 2,500 g. For each ChIP reaction, 60 μL of Magna ChIP™ Protein A+G Magnetic Beads (Merck Millipore) was used in accordance with the manufacturer's protocol. Before addition of the sheared chromatin to the beads, Triton X-100 and Na-deoxycholate was added to a final concentration of 10% each. 1% of the chromatin volume was used for input. At least two independent ChIP experiments were performed. Immunoprecipitated chromatin was deep-sequenced in the Genomics Unit of the Centre for Genomic Regulation (CRG, Barcelona, Spain) using the Illumina HiSeq 2500 system (Illumina). Briefly, library preparation included end-repair, generation of dA overhangs, adapter ligation, size selection and removal of non-ligated adapters by agarose gene electrophoresis and amplification (18 cycles) before loading the samples into the sequencer.