title: Illumina HiSeq 4000 paired end sequencing; Targeted DNA-seq of Human HepG2 cells following editing with CRISPR/Cas9
design: Targeted DNA-seq of Human HepG2 cells following editing with CRISPR/Cas9
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. sgRNA pools are described in the additional files associated with the submission Table_S1_sgrna_metadata.tsv and Table_S6_pools.tsv. HepG2 cells inducible for Cas9 expression were generated as previously described (Henser-Brownhill et al., 2017; 10.1080/15592294.2017.1395121). 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 extraction. Transduced cells were selected with 4ug/mL blasticidin, starting 24h after the first infection, and genomic DNA was extracted 5 days after infection (Qiagen 69506). DNA libraries enriched for the targeted sites were generated using the SureSelect Target enrichment kit (Agilent) following the manufacturer's instructions. Capture probes were designed to cover 2Kb regions centered on each target site.
attributes.tag: Experimental Factor: genotype
attributes.value: CRISPR/Cas9 mediated knockout with sgRNA pool 100-1