GSM5018597: AutophagyActivated-replicate1; Homo sapiens; OTHER
Genomic DNA was extracted using Blood & Cell Culture Midi kit (13343; QIAGEN). A total of 100 - 240 µg genomic DNA (varying between replicates) was extracted, and sgRNA sequences were amplified by PCR using Herculase II Fusion DNA polymerase (600675; Agilent Technologies). Next, the amplicons were subjected to the second PCR to attach Illumina adaptors and barcode samples. The primers used in the first PCR were as follows: F1, 5'-AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG-3', and R1, 5'-CTTTAGTTTGTATGTCTGTTGCTATTATGTCTACTATTCTTTCC-3'. The primers used in the second PCR were as follows: F2, 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-(variable-length sequence, up to 8 bp)-TCTTGTGGAAAGGACGAAACACCG-3'  R2, 5'-CAAGCAGAAGACGGCATACGAGAT-(8-bp barcode)-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTACTATTCTTTCCCCTGCACTGT-3'. DNA was sequenced on a HiSeq 2500 (Illumina) sequencer. After data acquisition, the number of reads that completely matched the GeCKO v2 library sequence was calculated.