Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
MEPCE

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
cell line
MDA-MB-231
shRNA status
shNC
chip antibody
Anti-MePCE, Origene, Catalog #TA590479, Lot #T01235A02

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed with 0.37% formaldehyde for 10 minutes at room temperature and quenched with 20 mM Glycine for 5 min. Cells were washed 2 times with cold PBS supplemented with EDTA-free Complete Protease Inhibitor cocktail from Roche (PIC), and scraped 2 times with 2.5 ml of cold PBS+PIC w/o EDTA. Pellets were suspended in 1.6 ml of RIPA buffer (50 mM Tris-HCl (pH 8), 150 mM NaCl, 2 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and PIC), sonicated in 15 ml conical tubes 3 times for 10 min at high 10 sec on/off cycles in a cooled Bioruptor and cleared by centrifugation for 15 min at 20,000 g at 4°C. The chromatin was quantified and the quality of DNA shearing was checked on a 1% agarose gel. The chromatin was diluted with RIPA Buffer to contain the equivalent of 10 ng/µl of DNA. 1 ml of chromatin corresponding to 10 µg of DNA was incubated with the appropriate antibodies O/N at 4°C, then with 25 µl of pre-washed Protein G Dynabeads® for 3 h at room temperature. The beads were washed for 5 min at RT with 1 ml of TSE-150 (0.1% SDS, 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl, pH 8), TSE-500 (0.1% SDS, 1% Triton X-100, 500 mM NaCl, 2 mM EDTA, 20 mM Tris-HCl, pH 8), LiCl buffer (0.25 M LiCl, 1% NP-40, 1% DOC, 1 mM EDTA, 10 mM Tris, pH 8) and TE (10 mM Tris-HCl, pH8, 0.1 mM EDTA). The immuno-complexes were eluted from the beads using a total of 125 µl of elution buffer (100 mM sodium bicarbonate, 1% SDS) for 30 min at 30°C and de-crosslinked overnight at 65°C. The ChIP samples were purified with the Qiaquick® PCR purification kit (Qiagen) and the DNA was eluted from the columns with 50 µl of water. 10 ng of DNA (size ~200 bp) from inputs and ChIP was used per ChIP-Seq library (Bioo ChIP-Seq kit) following manufacturers instructions. 8 barcoded libraries were sequenced in one lane with a HiSeq2000 instrument to obtain single-end, 36 bp reads.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
12602742
Reads aligned (%)
7.3
Duplicates removed (%)
84.0
Number of peaks
50 (qval < 1E-05)

hg38

Number of total reads
12602742
Reads aligned (%)
8.0
Duplicates removed (%)
82.7
Number of peaks
37 (qval < 1E-05)

Base call quality data from DBCLS SRA