Illumina HiSeq 2000 sequencing; Genome-wide mapping of HIST1H2ac binding sites in MCF-7 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
2016-05-19
ENA last update
2018-03-08
ENA-CHECKLIST
ERC000011
External Id
SAMEA3174169
INSDC center alias
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan
INSDC center name
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan
INSDC first public
2016-05-19T17:00:51Z
INSDC last update
2018-03-08T22:11:06Z
INSDC status
public
Submitter Id
E-MTAB-3160:H2ac_Rep2
broker name
ArrayExpress
cell line
MCF-7
common name
human
sample name
E-MTAB-3160:H2ac_Rep2
Sequenced DNA Library
library_name
H2ac_Rep2
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
MCF-7 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°C incubator with 5% CO2. Immunoprecipitation was performed according to the manufacturer’s protocol (Upstate Biotechnology, Inc., Lake Placid, NY) with slight modifications. MCF-7 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 anti-HA tag antibody (ab1424; 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°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).