GSM1868499: LNCaP SMARCA2 Vehicle ChIPSeq; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
SMARCA2
Cell type
Cell type Class
Prostate
Cell type
LNCAP
Primary Tissue
Prostate
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
prostate
cell type
prostate cancer cells
passages
58-65
cell line
LNCaP
chip antibody
SMARCA2 (Abcam, ab15597)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were grown to 70%-80% confluence, and crosslinked with 1% formaldehyde for 10 min at room temperature. After washing twice with cold PBS, cells were collected and resuspended in lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris (pH 8.0), 1x protease and phosphatase inhibitors). After sonication, the soluble chromatin was diluted in 1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris (pH 8.0), 1x protease and phosphatase inhibitors). Then samples were precleared and immunoprecipitated with the appropriate antibodies overnight at 4°C. The next day, protein A or G beads blocked with BSA were added for 2 h at 4°C, and then washed sequentially for 10 min with TSE I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8.0), 150 mM NaCl), TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8.0), 500 mM NaCl), buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris (pH 8.0), and TE buffer (10 mM Tris (pH 8.0), 1mM EDTA (pH 8.0)). DNA was eluted in Elution buffer (1% SDS, 0.1M NaHCO3) at room temperatue for 30 min. The crosslinking was reversed by incubation for 16 h at 65°C. During the purification, ChIP DNA was treated with DNase-free RNase for 30 min at 37°C. Libraries were prepared according to NEBNext ChIP-Seq Library Prep Master Mix Set (E6240L). 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 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 10 or 14 cycles and library fragments of ~300 bp (insert plus adaptor and PCR primer sequences) were band isolated from an E-gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq5000 following the manufacturer's protocols.