NGS library preparation and sequencing for CRISPR/CAS9 outcomes

Gene-specific primers were used to amplify a region flanking the gRNA target site; with tags 5′-CGCTCTTCCGATCTCTG-3′ and 5′-TGCTCTTCCGATCTGAC-3′ appended to the 5′ side of the gene-specific portion for forward and reverse primers, respectively. These amplicons were used as a template for the second PCR to add the Ion Xpress barcode adapters. Equimolar concentrations from each amplicon were pooled to make the final sequencing library. The library was diluted to 100 pM and sequencing template was prepared using the Ion PGM Hi-Q OT2 Kit on the Ion One Touch 2 instrument according to manufacturer's instructions. Templated Ion Sphere Particles were enriched on an Ion One Touch ES and sequencing was performed on an Ion Torrent PGM with the Ion PGM Hi-Q Sequencing Kit. Demultiplexing of barcoded samples was performed using Torrent Suite Software 5.0.4 and reads were trimmed using Cutadapt.¹³ CRISPR-Cas9 outcomes were quantified using CRISPResso computational pipeline.¹⁴