GSM9571429: LcV2-Lockwood23-N2; synthetic construct; OTHER
Plasmid DNA was extracted from bacterial colonies via Qiagen Maxiprep Preliminary library sequencing was performed by setting up x4 50uL PCR reactions (PCR1) using Q5® Hot Start High-Fidelity 2X Master Mix (NEB, catalogue number M0494L) using 50ng of plasmid library per reaction. PCR was performed per manufacturer's instructions for 12 cycles. Forward primer mix for PCR1 was an equimolar mix of 8 primers with varying numbers of degenerate bases (S1-8). Primer sequences are outlined in Supplementary Table 1 (Library-NGS-S1-8-F, Lenti-CRISPR-V2-NGS-R). PCR1 products were pooled and cleaned up using AmpureXP beads (Beckman Coulter, catalogue number A63880) at a ratio of 0.8X. Beads were washed twice with 80% ethanol before being eluted in 50uL IDTE. Eluate was cleaned up again following the same protocol with 0.8X bead ratio, and entire product was eluted into 50uL IDTE. Final product was used as a template for x4 50uL PCR reactions using Q5® Hot Start High-Fidelity 2X Master Mix for barcoding and addition of Illumina sequencing adapters (PCR2). PCRs were run according to manufacturer's instructions for 10 cycles. PCR2 primer sequences are outlined in Supplementary Table 1. PCR2 products were cleaned up using the same bead protocol described, however only a single binding and elution was performed. Samples were sent to the UBC Sequencing Consortium (USC) for quality check, pooling, and sequencing on Illumina MiSeq using the Nano v2 kit with 20% PhiX spike in. Samples were run using a 2x150bp program, though only Read1 was utilized for sgRNA alignment. sgRNA counts were generated using R, all adapter trimming and sorting was performed by the USC.