NGS CRISPR off-target hMSC_NT #2
Customized PCR primers for the on-target and off-target sites to amplify the sgRNA target region were ordered to IDT with rhAmpSeq index sequences. Each locus was individually amplified using genomic DNA extracted from the relevant cells. First PCR was performed rhAmpSeq Library Kit (IDT) under the following conditions: 95C for 10 min, 10 cycles of 95C for 15 s and 61C for 4 min, and 99.5C for 15 min. PCR products were subsequently equally pooled and purified with Agencourt AMPure XP beads (Beckman). The PCR products were then subjected to a second PCR with Illumina indexed adaptors primers under the following PCR conditions: 95C for 3 min, 18 cycles of 95C for 15 s, 60C for 30 s and 72C for 30 s, and 72C for 1 min. Second PCR products were purified with Agencourt AMPure XP beads (Beckman) at a PCR product:bead ratio of 1:1. The purified libraries were quantified and sequenced using a 300-bp paired-end cycle on the Illumina MiSeq sequencing platform.