Fs dna library prep kit

To assay off-target sites, HEK293T cells were nucleofected with precomplexed RNP consisting of 104 pmol of the indicated protein and 120 pmol sgRNA using the Lonza 4D electroporation system. In parallel, cells were cotransfected with 5 pmol annealed double stranded oligodeoxynucleotide (dsODN) as described.^{39 (link)} After 72 h, cells were trypsinized and genomic DNA (gDNA) extracted using the PureLink gDNA extraction kit and quantified. Four hundred nanograms of high-molecular-weight gDNA was fragmented, end-repaired, and ligated using the NEB FS DNA Library Prep Kit. Fragments between 350 and 600 bp were amplified to enrich for dsODN-proximal regions using dsODN-specific primers in both the positive and negative orientations. Resulting libraries were amplified for NGS on Illumina MiSeq and sequenced as 2 × 150 paired end reads. Reads were analyzed using a modified guideseq software package (adapted from http://github.com/aryeelab/guideseq). The Venn diagrams for Supplementary Figure S12 were visualized with BioVenn.^{40 (link)}

HEK293T cells were nucleofected with precomplexed RNP consisting of either 12 pmol Cas9 and 60 pmol sgRNA, 104 pmol MG3-6 and 120 pmol sgRNA, or 120 pmol MG29-1 and 120 pmol sgRNA, using the Lonza 4D electroporation system. In parallel, cells were cotransfected with 50 pmol annealed dsODN. After 72 h, cells were trypsinized and gDNA extracted using the Kingfisher MagMAX DNA Multi-Sample Ultra 2.0 Kit (Catalog No. A36570). Four hundred nanograms of high molecular weight gDNA was fragmented, end-repaired, and ligated using the NEB FS DNA Library Prep Kit (Catalog No. E7805). Fragments between 350 and 600 bp in length were amplified to enrich for dsODN-proximal regions using dsODN-specific primers in both the positive and negative orientations. Resulting libraries were amplified for next generation sequencing (NGS) on Illumina Miseq.