Amplicon sequencing for CRISPR guide library: sorted population
Genomic DNA (gDNA) was extracted from cell pellets of 40-50 million cells using the QIAamp DNA Blood Maxiprep Kit (Qiagen # 51192) according to manufacturers instructions with minor modifications: QIAGEN Protease was replaced with 500 L of a 10 mg/mL solution of ProteinaseK (MilliporeSigma # 3115879001) in water; cells were lysed overnight; centrifugation steps after Buffer AW1 and AW2 were performed for 2 minutes and 5 minutes, respectively;  1 mL of water preheated to 70 C was used to elute gDNA (5-minute incubation), followed by centrifugation for 5 minutes. gDNA was quantified using the Qubit dsDNA HS Assay kit (Thermo Fisher Scientific #Q32851).All PCR reactions were performed in 50 L reactions using ExTaq Polymerase (Takara Bio #RR001B) using the following primers:Forward: 5- AATGATACGGCGACCACCGAGATCTACACCCCACTGACGGGCACCGGA - 3Reverse: 5- CAAGCAGAAGACGGCATACGAGATCnnnnnnTTTCTTGGGTAGTTTGCAGTTTT - 3Where nnnnnn denotes the barcode used for multiplexing.For all samples, 1, 3, or 6 g of gDNA was initially amplified for 28 cycles in 50 L test PCR reactions. Subsequently, an additional 23.33 reactions were performed using 6 g per reaction (140 g gDNA). Reactions were pooled and 100 L of each reaction was purified using Select-a-Size DNA Clean and Concentrator (Zymo Research #D4080), eluted with 15 L water, and quantified using the Qubit dsDNA HS Assay kit before sequencing for 50 cycles on an Illumina Hiseq 2500 using the following primers:Read 1 sequencing primer: 5- GTTGATAACGGACTAGCCTTATTTAAACTTGCTATGCTGTTTCCAGCATAGCTCTTAAAC - 3Index sequencing primer: 5- TTTCAAGTTACGGTAAGCATATGATAGTCCATTTTAAAACATAATTTTAAAACTGCAAACTACCCAAGAAA - 3