MRP_Enriched_sgRNA_Library
Enriched sgRNA library after the third round of forward genetic screen in the "Green" Fireworks cell line. Genomic DNA was isolated from 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 GF4 (5-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNTGTTCTTGTGGAAAGGACGAAACACCG-3) and GRfixed (5-CAAGCAGAAGACGGCATACGagatACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAAC-3).