Next-Generation Sequencing (NGS) and pooled screen analysis
Plasmid DNA was isolated using a Plasmid Maxiprep Kit (Biomed).
Integrated guide RNA target pair sequences were PCR-amplified using Q5
polymerase (NEB) for frequency analysis. PCR products were purified
using a QIAquick Purification Kit (QIAGEN). For each sample, 1 microgram
of purified PCR amplicons were used as a library template. Sequencing
libraries were obtained from the replicates using a NEBNext Ultra II RNA
Kit (NEB)and purified with SPRI beads(Beckman). The sequencing libraries
were prepared following the manufacturer’s protocol(Illumina).Library
size and purity was verified by Agilent 2100(Agilent) before sequencing
on a Nova seq (Illumina) using a Reagent Kit S2 (Novaseq) (2 × 150 bp).