Optimized CROP-seq gRNA Library Preparation

CROP-seq-opti gRNA sequencing libraries were prepared as previously described [113 (link)]. Briefly, 100ng of each gDNA sample was PCR amplified in triplicate with Q5 High-Fidelity DNA Polymerase (NEB #M0494S) with 500nM primers (S4 Table) flanking the guide sequence cassette and including Illumina adaptor sequences and sample index sequences (98°C x 30s, 98°C x 10s, 72°C x 45s, 25 cycles). PCR products were purified using 2.0X AMPure XP magnetic beads (Beckman Coulter #A63881) according to manufacturer’s protocol. Sequencing libraries were quantified with the KAPA Library Quantification Kit (Roche #07960140001), pooled, and sequenced in multiplex on the Illumina NextSeq 500 platform using a 150-cycle mid output kit (Illumina # 20024904) with read configuration of 167 bases (read 1) and 8 bases (i7 index).