title: Illumina MiSeq paired end sequencing; Targeted DNA-seq of Human HepG2 cells following editing with CRISPR/Cas9 upon chromatin modulation with TSA and EZH2i
design: Targeted DNA-seq of Human HepG2 cells following editing with CRISPR/Cas9 upon chromatin modulation with TSA and EZH2i
library_construction_protocol: 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.
attributes.tag: Experimental Factor: compound
attributes.value: GSK126
attributes.tag: Experimental Factor: dose
attributes.value: 3
attributes.tag: Experimental Factor: genotype
attributes.value: CRISPR/Cas9 mediated double knockout of ACTL6A.5 and MBD3L1.6 sites