Illumina Genome Analyzer IIx sequencing; Pol III ChIP-seq of liver cancer cell lines
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
RNA polymerase
Antigen
RNA polymerase II
Cell type
Cell type Class
Liver
Cell type
Hep G2
Primary Tissue
Liver
Tissue Diagnosis
Carcinoma Hepatocellular
Attributes by original data submitter
Sample
ENA first public
2016-04-18
ENA last update
2018-03-09
ENA-CHECKLIST
ERC000011
External Id
SAMEA3662505
INSDC center alias
Cambridge Institute University of Cambridge Cancer Research UK
INSDC center name
Cambridge Institute University of Cambridge Cancer Research UK
INSDC first public
2016-04-18T17:01:17Z
INSDC last update
2018-03-09T09:25:53Z
INSDC status
public
Submitter Id
E-MTAB-4046:HepG2_rep1
broker name
ArrayExpress
cell line
HepG2
cell type
hepatocyte
common name
human
disease
hepatocellular carcinoma
immunoprecipitate
Pol III (RPC1/155)
sample name
E-MTAB-4046:HepG2_rep1
component_organism
Homo sapiens
component_organism
Homo sapiens
Sequenced DNA Library
library_name
HepG2_rep1
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Liver hepatocellular carcinomas cell lines HepG2 and Huh7 from human and Hepa1-6 and Hepa-1c1c7 from mouse were grown to 80% confluency in DMEM (Sigma) supplemented with 10% foetal bovine serum (FBS) and antibiotics (100 g/l penicillin and 100 g/ml streptomycin) at 37C and 5% CO2. Cells were fixed in 1% formaldehyde (v/v). After 20 minutes, the cross-linking reaction was quenched with 1/20 volume of 2.5 M glycine to neutralize the formaldehyde. Cells were resuspended in 10ml Lysis Buffer 1 (50mM Hepes-KOH [pH 7.5], 140mM 5M NaCl,1nM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100, Proteinase Inhibitor). Rotated at 4oC for 10 min. Centrifuged at 2,000g 5 min at 4oC. After removal of the supernatant the pellet was resuspended in 10ml Lysis Buffer 2 (10mM Tris-HCL [pH 8.0], 200mM NaCL, 1mM EDTA, 1mM EGTA, Proteinase Inhibitor). Rotated at 4oC for 5 min. Centrifuge at 2,000g 5 min at 4oC. After removal of the supernatant the pellet was resuspended in 3ml Lysis Buffer 3 (10mM Tris-HCL [pH 8.0], 100mM NaCL, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, Proteinase Inhibitor). Suspension was sonicated (8-12 cycles of 30 sec on and 60sec off) with a micro tip attached to Misonix 3000 sonicator. Tubes were kept on ice water. 30 l 10% Triton X-100 were added to sonicated lysate. Centrifuge at 20,000g for 10 min at 4oC. Immunoprecipitated DNA was end-repaired, A-tailed, ligated to Illumina sequencing adapters, amplified by 18 cycles of PCR and size selected (200300 bp).