Amplicon gRNA seq of FlpIn Trex 293 cells treated with dCas13d/gRNA
For each screen replicate, the samples processed for gRNA sequencing included unsorted cells and top and bottom bins. The genomic DNA was extracted using Monarch Genomic DNA purification kit (NEB T3010). Two rounds of amplification for gRNA from the genomic DNA were performed. The first PCR amplified a region (547 bp) containing the gRNA. A minimum of 6 PCR reactions were set up per sample, and 200 ng of genomic DNA was used per reaction using NEB Q5 Hot Start High-Fidelity master mix (NEB M0494) with PCR1 conditions: 98C for 30s, 24 (98C for 10s, 66C for 30s, 72C for 45s), 72C for 5min). The PCR reactions for each sample were pooled and cleaned up with the QIAquick PCR purification kit (Qiagen 28104). Using the pooled PCR1 products, the second PCR reaction per sample was performed. PCR2 adds the sample-specific barcode, Illumina sequencing adapters, and stagger sequences to prevent sequencing issues associated with lack of sequence diversity (i.e., region of direct repeat). Again, NEB Q5 Hot Start High-Fidelity master mix was used with PCR2 conditions: 98C for 30s, 16 (98C for 10s, 66C for 30s, 72C for 45s), 72C for 5min). The products from PCR2 were purified using gel extraction (QIAquick gel extraction 28706). The individual samples libraries were quantified using the KAPA qPCR Illumina library quantification kit (Roche 07960140001). Libraries were pooled in equal ratios and sequenced on a NextSeq 550 using a Mid Output kit v2.5 with 1x150 cycles.