DNA-seq (low-coverage WGS) for validation of CRISPR-Cas9-edited mESCs
DNA-seq (low-coverage WGS) for validation of CRISPR-Cas9-edited mESCs
Mouse embryonic stem cells (129XC57BL/6J) were cultured in ESC medium containing Knockout-DMEM (Thermo Fisher Scientific) supplemented with 15% EmbryoMax fetal bovine serum (ES-FBS Merck-Millipore) and 20 ng/ml leukemia inhibitory factor (LIF, produced by Protein Expression and Purification Facility at EMBL Heidelberg), 1% non-essential amino acids (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 1% Pen/Strep (Thermo Fisher Scientific), 1% of 55 mM beta-Mercaptoethanol solution (Merck Millipore). Upon thawing, cells were cultured on a layer of mouse embryonic fibroblasts (MEFs) and afterwards passaged on 0.1% gelatine-coated culture dishes, without feeder cells. Cells were maintained at 37°C with 5% CO2 and routinely tested for mycoplasma. The medium was changed daily, and cells were passed every two days. Genomic DNA was extracted from ~10 million cells using the Gentra Puregene Cell Kit (Qiagen). Genomic DNA was extracted from ~10 million cells using the Gentra Puregene Cell Kit (Qiagen) and sonicated at 4°C using a Bioruptor Pico sonication device (Diagenode) for 10 cycles (30s ON/30s OFF) to obtain fragments of ~200 bp. The fragmentation pattern was assessed via agarose gel electrophoresis and 0.5-1 μg of fragmented gDNA was used to prepare sequencing libraries using the NEBNext Ultra II DNA Library Preparation Kit (New England Biolabs), according to manufacturer's instructions. Libraries were then quantified using the Qubit fluorometer (Thermo Fisher Scientific) and the quality was assessed on a Bioanalyzer using the Agilent High Sensitivity DNA Kit.