Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
BRD4

Cell type

Cell type Class
Breast
Cell type
SUM 159PT
Primary Tissue
Breast
Tissue Diagnosis
Anaplastic Carcinoma

Attributes by original data submitter

Sample

source_name
breast cancer cells
cell line
SUM159R
antibody
anti-BRD4 (Bethyl, A301-985A)
treatment
DMSO
replicate
1

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells (1x107) were grown in SUM Medium. The media were then removed and replaced with media containing 1% formaldehyde (EM grade; tebu-bio) and crosslinked for 8 min. Crosslinking was quenched by adding glycine to a final concentration of 0.2 M. The cells were washed with ice-cold PBS, harvested in PBS, and the cell pellet was washed with PBS. The nuclear fraction was extracted by first resuspending the pellet in 10 ml of LB1 buffer (50mM HEPES-KOH [pH 7.5], 140mMNaCl, 1mMEDTA, 10% glycerol, 0.5% NP-40 or Igepal CA-630, and 0.25% Triton X-100) for 10 min at 4C. Cells were pelleted, resuspended in 10 ml of LB2 buffer (10 mM Tris-HCL [pH 8.0], 200 mM NaCl, 1 mM EDTA, and 0.5 mM EGTA), and mixed for 5 min. Cells were pelleted and resuspended in 1ml LB3 buffer (10 mM Tris-HCl [pH 7.4], 1 mM EDTA, 0.1%SDS, 1%Triton X-100, 0.1% Na-deoxycholate, 1Mm DTT, 0.25% N-lauroylsarcosine, protease inhibitors and phosphatase inhibitors) and sonicated in a covaris sonicator for 10 min. A total of 30 l of 5M Nacl was added, and lysate was centrifuged for 10 min at 20,000 rcf to purify the debris. The supernatant was then incubated with 50 l of magnetic beads (Life Technologies) prebound with 20 g BRD4 antibody (Bethyl, A301-985A), and immunoprecipitation (IP) was conducted overnight in the cold room. The beads were washed ten times in 1 ml of RIPA buffer and twice in 100mM ammonium hydrogen carbonate (AMBIC) solution. DNA was eluted in elution buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS). Cross-links were reversed overnight at 65C. RNA and protein were digested with 0.2 mg/mL RNase A for 2 hr followed by 0.2 mg/mL Proteinase K for 1 hr. DNA was purified with phenol chloroform extraction and ethanol precipitation. ChIP-seq libraries were prepared using the Rubicon ThruPLEX DNA-seq Kit from 1 ng of purified ChIP DNA or input DNA according to the manufacturer's protocol.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
57252546
Reads aligned (%)
49.1
Duplicates removed (%)
45.6
Number of peaks
1440 (qval < 1E-05)

hg38

Number of total reads
57252546
Reads aligned (%)
52.0
Duplicates removed (%)
43.1
Number of peaks
2239 (qval < 1E-05)

Base call quality data from DBCLS SRA