DNA-seq (low-coverage WGS) for validation of CRISPR-Cas9-edited mESCs

Mouse embryonic stem cells (i.e., mESCs; line ESC 129-B13) were genetically modified using CRISPR-Cas9 to mutate the H3f3b locus, in order to carry homozygous lysine-to-alanine substitution of residues K9, K27 or K79. Two control mESC lines carrying knock-out of the H3f3a gene were used as background for the editing. Low-coverage whole-genome sequencing (~0.8/0.9X) was performed to assess the chromosomal integrity of the edited lines. Genomic DNA was extracted from each mutant and control mESC line and sonicated to obtain fragments of ~150-200bp on average. Fragmented DNA (~0.5-1 ug) was used for library preparation using the NEBNext Ultra II DNA library preparation kit (New England Biolabs). Sequencing was performed on a NextSeq500 platform in single-end mode (75bp reads).