NextSeq 500 sequencing; Histone ChIPSeq of H3K79me2 before and after EPZ004777 treatment 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
2019-02-08
ENA last update
2018-06-11
ENA-CHECKLIST
ERC000011
External Id
SAMEA4722381
INSDC center alias
Laboratory of Meolcularc Medicine and Genomics
INSDC center name
Laboratory of Meolcularc Medicine and Genomics
INSDC first public
2019-02-08T17:04:05Z
INSDC last update
2018-06-11T15:19:11Z
INSDC status
public
Submitter Id
E-MTAB-6889:Sample 3
broker name
ArrayExpress
cell line
MCF-7
cell type
mammary epithelial cell
common name
human
disease
invasive ductal carcinoma
metastatic site
pleural effusion
organism part
mammary gland
sample name
E-MTAB-6889:Sample 3
Sequenced DNA Library
library_name
Sample 3_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ER-alpha positive BC cells MCF-7 (ATCC HTB-22) were propagated in Dulbecco's modified Eagle medium (DMEM; Sigma-Aldrich) supplemented with 10% FBS (HyClone) and antibiotics: 100 U/ml penicillin, 100 mg/ml streptomycin, 250 ng/ml amphotericin-B For histone extraction 100.000 cells/well were seeded in 35 mm plates were incubated with increasing concentrations of Dot1L inhibitors: EPZ004777 (S7353, Sellekchem), EPZ5676 (S7062 Sellekchem) and SGC0946 (S7079, Sellekchem) and their vehicle for 6 days. For this experiment we use only EPZ004777 (S7353, Sellekchem) at 6.4 microM. Cells were harvested, washed twice with ice-cold PBS and the pellets were resuspended in Triton Extraction Buffer (TEB: PBS containing 0.5% Triton X 100 (v/v), 2 mM phenylmethylsulfonyl fluoride (PMSF). Cells were lysed on ice for 10 min and nuclear separation was obtained by centrifugation at 6,500 x g for 10 min at 4°C. Then, the nuclei were resuspended in 0.2 N HCl before acid extraction of histones over night at 4°C. The histone content was determined using the Bradford assay and was analyzed by Western blot procedure. Antibody used:: Rb pAb Histone3 (di methyl k79); catalogue no: ab3594; Company: abcam After binding, beads washing, elution, reverse crosslinking and DNA extraction were performed as described in Tarallo et al, 2014. Three biological replicates were performed for each experimental condition. Then, 1µl aliquot of obtained DNA was used to asses size distribution by Agilent Genomic DNA ScreenTape & Reagents using an Agilent 4200 TapeStation System (Agilent Technologies). The concentration of each DNA sample was determined by using Quant-IT DNA Assay Kit-High Sensitivity and a Qubit Fluorometer (Life Technologies). 10 ng of each purified ChIP DNA were used for sequencing libraries preparation with TruSeq ChIP Sample Prep Kit (Illumina Inc.).