Illumina HiSeq 2000 sequencing; ChIP-Seq for RNA Polymerase II in MCF7 breast cancer cells
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
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
ENA first public
2015-02-25
ENA last update
2018-03-08
ENA-CHECKLIST
ERC000011
External Id
SAMEA2220301
INSDC center alias
National Yang-Ming University
INSDC center name
National Yang-Ming University
INSDC first public
2015-02-25T17:03:15Z
INSDC last update
2018-03-08T16:47:53Z
INSDC status
public
Submitter Id
E-MTAB-1952:MCF7 cells
broker name
ArrayExpress
cell line
MCF7
cell type
human breast cancer cell
common name
human
sample name
E-MTAB-1952:MCF7 cells
sex
female
Sequenced DNA Library
library_name
extract 1
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
MCF7 cells were cultured in RPMI1640 medium (GIBCO/BRL) supplemented with 10% (v/v) fetal bovine serum (GIBCO/BRL), 2.0 g/L sodium bicarbonate, and were incubated in a humidified 37 degrees C incubator with 5% CO2. Immunoprecipitation was performed according to the manufacturer's protocol (Upstate Biotechnology, Inc., Lake Placid, NY) with slight modifications. MCF7 cells were fixed for 10 min with 1% of formaldehyde at room temperature, and then quenched with a final of 1M of glycine. The cells were lysed and the chromatin was sonicated to 200-500 bp fragments with a Bioruptor (Diagenode, Sparta, NJ). Chromatin was immunoprecipitated by using RNA polymerase II (ab5408-100; Abcam) and Protein A Agarose beads. The beads were washed once with each washing buffer, including low salt immune complex wash buffer, high salt immune complex wash buffer, and LiCl immune complex wash buffer, and twice with 1X TE buffer. Precipitates were eluted with 1% of SDS and 100 mM of NaHCO3. The samples were heated at 65 degrees C for 6 hr in order to reverse cross-link, extracted with phenol/chloroform, ethanol-precipitated. The sequencing libraries were constructed from immunoprecipitated and input DNA using TruSeq ChIP Sample Preparation Kit (Illumina Inc., USA) according to the manufacturer's instruction. The fragmented DNA was end repaired following by addition 3'-A to the ends and ligation of adapters. The adapter-ligated DNA library was sized-selected (300-500 bp) on a 2% agarose gel and amplified by PCR for 16 cycles with the use of KAPA HiFi DNA Polymerase (Kapa Biosystems).