Enriched sgRNA library
Enriched sgRNA library after the first round of forward genetic screen in the "Red" Mirror cell line. Genomic DNA was isolated from 25,000 FACS-isolated cells using phenol extraction. Sequences of guide RNAs were amplified from genomic DNA in two steps. First, a linear sgRNA amplification was performed using Herculase II DNA polymerase using 13 thermal cycles with a single sgRNA promoter-specific primer, RandomF (5-TAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG-3), and the following cycling parameters: 96°C 20s, 63°C 1min, 72°C 90s. Then, the second PCR primer, RandomR (5-ACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3), was added to the reaction and a regular PCR was performed for 35 cycles as follows: 96°C 3min; 35 cycles of 96°C 20s, 63°C 1min, 72°C 45s; 72°C 10min; 4°C. Then, illumina sequencing adaptors were added by PCR using primers GF5 (5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNACGTCTTGTGGAAAGGACGAAACACCG-3) and GR (5-CAAGCAGAAGACGGCATACGACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAAC-3).