Pool1
Homo sapiens
Protocols: HepG2 cells were cultured in Minimum Essential Media (MEM) with 10% FBS. All media was supplemented with 2mM L-glutamine, 100U/ml penicillin, and 100μg/ml streptomycin. HepG2 cells inducible for Cas9 expression were generated as previously described (Henser-Brownhill et al., 2017). HepG2 cells were treated with the HDAC inhibitor Trichostatin A, which induces histone hyperacetylation, and the EZH2 inhibitor GSK126, which globally reduces H3K27Me3 levels. Cells pre-treated with TSA (11nM or 100nM) or EZH2 (0.3 µM and 3 µM) for 5 days were infected with sgRNAs targeting the ACTL6A.5, ASF1B.7, BRD2.7, MBD3L1.6, MSH6.2 and SMARCD2.1 sites. Cas9 induction was induced with 1ug/mL doxycycline 1 day prior to infection with the sgRNAs and sustained until cells were harvested for genomic DNA. Genomic DNA was extracted 5 days after infection (Qiagen 69506). For the first PCR, 150ng of the corresponding gDNA were amplified for 20-22 cycles using the Herculase II polymerase kit yielding products of ~500bp. Next, PCR products were purified as per manufacturer's instructions (Qiagen 28106) and 1µl of the resulting product was used as a template for the second nested PCR reaction in which primers containing barcodes and adapters for the sequencing reaction were added.
ENA-FIRST-PUBLIC
2018-12-13T17:03:14Z
ENA-LAST-UPDATE
2018-07-30T15:26:10Z
External Id
SAMEA4809344
INSDC center name
The Francis Crick Institute
INSDC first public
2018-12-13T17:03:14Z
INSDC last update
2018-07-30T15:26:10Z
INSDC status
public
Submitter Id
E-MTAB-7091:Pool1
broker name
ArrayExpress
cell line
HepG2
cell type
epithelial cell
common name
human
disease
hepatocellular carcinoma
genotype
CRISPR/Cas9 mediated double knockout of ACTL6A.5 and MBD3L1.6 sites
organism part
liver
sample name
E-MTAB-7091:Pool1
scientific_name
Homo sapiens