Genome-wide CRISPR Screening Library Preparation

To prepare libraries for sequencing, 430 μg of extracted genomic DNA per sample were amplified and prepared for sequencing using Phusion Flash high-fidelity master mix (Thermo Fisher F548L) according to a previously described protocol (Chen et al. 2015 (link)). A two-step PCR reaction was employed to first amplify the sgRNA cassette and maintain library complexity (12 cycles), and a second PCR reaction was performed to add barcodes and Illumina adapters (16 cycles). Reactions were pooled after each PCR. Following the second PCR, reaction products were run on a 2.5% agarose gel and the DNA purified using a gel extraction kit (PureLink gel extraction kit; Invitrogen K210012). Once purified, the concentration of individual libraries was quantified using the Kapa Biosystems library quantification kit (Roche KK4824) according to kit instructions. Libraries were sequenced using an Illumina HiSeq 2500. The sequencing results were analyzed using the MAGeCK-VISPR pipeline (Li et al. 2015 (link)). For CRISPR score analyses of aligned and normalized read counts, the following formula was used: CRISPR score = log₂ (final sgRNA abundance/initial sgRNA abundance) (Wang et al. 2015 (link)).