Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
BRD3

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
BRD3_ChIP-seq_MCF-7_E2
cell line
MCF-7
treatment
E2 (100 nM), 45 min
chip antibody
BRD3
chip antibody vendor
Bethyl Laboratories

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP was performed as previously described with a few modifications. MCF-7 cells were plated at a density of 2.5 x 106 cells in a 15 cm diameter dish, grown for three days in estrogen-free medium, and treated as described above (± 45 minutes with 100 nM E2; ± 3 hour pretreatment with 500 nM JQ1). The cells were cross-linked with 1% formaldehyde for 10 min at 37oC and quenched in 125 mM glycine for 5 min at 4oC. The cells were then collected and lysed in Farnham lysis buffer [5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40, 1 mM DTT, and 1x complete protease inhibitor cocktail (Roche, 11697498001)]. For acetyl-histone H4 ChIP assays, 10 mM sodium butyrate was added to all buffers to prevent deacetylation of the histones. The crude nuclear pellet was collected by centrifugation, resuspended in lysis buffer [Tris-HCl pH 7.9, 1% SDS, 10 mM EDTA, 50 mM, 1 mM DTT, and 1x complete protease inhibitor cocktail], and incubated on ice for 10 minutes. The chromatin was sheared by sonication at 4¡C using a Bioruptor UC200 at the highest setting for five 5-minute cycles of 30 seconds on and 60 seconds off to generate chromatin fragments of ~200-400 bp in length. The soluble chromatin was diluted 1:10 with dilution buffer [20 mM Tris-HCl pH 7.9, 0.5% Triton X-100, 2 mM EDTA, 150 mM NaCl, 1 mM DTT and 1x complete protease inhibitor cocktail] and pre-cleared with protein A agarose beads. Five percent of the material was removed and saved as input, and the rest of the pre-cleared supernatant was incubated overnight at 4oC with the specified antibodies of interest or without antibody as a control (each 15 cm diameter dish yielded two immunoprecipitations) with continuous mixing. After the incubation, the immune complexes were collected by adding protein A agarose beads and incubating for 2 hours at 4°C. The immunoprecipitated material was washed once with low salt wash buffer [20 mM Tris-HCl pH 7.9, 2 mM EDTA, 125 mM NaCl, 0.05% SDS, 1% Triton X-100, and 1x complete protease inhibitor cocktail], once with high-salt wash buffer [20 mM Tris-HCl pH 7.9, 2 mM EDTA, 500 mM NaCl, 0.05% SDS, 1% Triton X-100, and 1x complete protease inhibitor cocktail], once with LiCl wash buffer [10 mM Tris-HCl pH 7.9, 1 mM EDTA, 250 mM LiCl, 1% NP-40, 1% sodium deoxycholate, and 1x complete protease inhibitor cocktail], and twice with Tris-EDTA (TE) containing 1x complete protease inhibitor cocktail. The immunoprecipitated material was eluted at room temperature in elution buffer [100 mM NaHCO3, 1% SDS], and reverse crosslinked by adding 100 mM NaCl and incubating at 65oC overnight. The eluted material was then digested with proteinase K and RNase A to remove protein and RNA, respectively, and the enriched genomic DNA was extracted with phenol:chloroform:isoamyl alcohol, followed by isopropanol precipitation. The ChIPed DNA was dissolved in water and purified again using AMPure Beads XP. ChIP-seq libraries were generated using two biological replicates the ChIPed DNA described above for each condition. The DNA was purified using a MiniElute PCR Purification Kit (Qiagen, 28004). After purification, 50 ng of ChIPed DNA for each condition was used to generate libraries for deep sequencing, as previously described {Quail, 2008 #8;Quail, 2008 #8;Franco, 2015 #9}, with some modifications. Briefly, the DNA was end-repaired and a single ÒAÓ-base overhang was added using the Klenow fragment of E. coli DNA polymerase. The A-modified DNA was ligated to Illumina sequencing adaptors using the Illumina TruSeq DNA Sample Prep Kit. The ligated DNA (300-500 bp) was size-selected by agarose gel electrophoresis and purified using a QIAquick Gel Extraction kit (Qiagen, 28704). The size-selected fragments were PCR amplified using Illumina TruSeq P5 and P7 PCR primers, and purified using AMPure beads (Beckman Coulter, A63881). After quality control analyses, the libraries were sequenced on an Illumina HiSeq 2000 (single-end sequencing, 50 nt).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
49268922
Reads aligned (%)
33.8
Duplicates removed (%)
62.9
Number of peaks
1052 (qval < 1E-05)

hg38

Number of total reads
49268922
Reads aligned (%)
35.6
Duplicates removed (%)
61.2
Number of peaks
1134 (qval < 1E-05)

Base call quality data from DBCLS SRA