gRNA insert of CRISPR KO library in CHO cells undergoing selection in media w/ or w/out glutamine; replicate 2 of cells after 14 days of growth in media w/out glutamine
50 uL PCR reactions with 3 ug input gDNA per reaction were run using Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) (95C for 4 min; 30 times: 98C for 45s, 60C for 30 s, 72C for 1 min; 72C for 7 min) using primers flanking the gRNA insert containing overhang sequenced compatible with Illumina Nextera XT indexing and 8 random nucleotides to increase the diversity of the sequences (LIB_8xN_NGS_FWD and LIB_8xN_NGS_REV listed in Supplemental Table S3). Double size selection was performed using Agencourt AMPure XP beads (Beckman Coulter) to exclude primer dimers and genomic DNA. The amplicons were indexed using Nextera XT Index Kit v2 (Illumina) sequence adapters using KAPA HiFi HotStart ReadyMix (KAPA Biosystems) (95C for 3 min; 8 times: 95C for 30s, 55C for 30 s, 72C for 30 s; 72C for 5 min) and subjected to a second round of bead-based size exclusion. The resulting library was quantified with Qubit using the dsDNA HS Assay Kit (Thermo Fisher Scientific) and the fragment size was determined using a 2100 Bioanalyzer Instrument (Agilent) before running the samples on a NextSeq 500 sequencer (Illumina).
t14.minusgln.r2