GSM7764465: CPEB2.PAS.Sorted.replicate 4; Homo sapiens; OTHER
Genomic DNA from unsorted and sorted (mCherry/GFP ratio increased) cells extracted by using Wizard Genomic DNA Purification Kit, and gDNA was PCR amplified. Sequencing libraries were prepared from total of 50 µg (approximately 100-fold coverage of the TKOv3 library) of the extracted genomic DNA in two PCR steps. For the first PCR, a total of 14 PCR reactions were carried out with each containing 3.5 µg (a total of 50 µg) of genomic DNA in 50 μl reaction with 2× NEBNext Ultra II Q5 Master Mix (NEB, catalogue number M0544L) to amplify the gRNA inserts using the forward primer v2.1.F1 GAGGGCCTATTTCCCATGATTC, and reverse primer v2.1.R1 GTTGCGAAAAAGAACGTTCACGG. For the second PCR, the gRNA was amplified using 5 μl of the pooled first PCR products as a template in a 50 μl reaction with NEBNext Ultra II Q5 Master Mix by using the primers harboring Illumina TruSeq adapters i5 (with staggered sequences) and i7 barcodes. The resulting libraries were sequenced on an Illumina HiSeq2500 to a depth of 10 to 20 millions, using single-read sequencing and completed with standard primers for dual indexing with HiSeq SBS Kit v4 reagents. The first 21 cycles of sequencing were dark cycles, or base additions without imaging. The actual 36-base read begins after the dark cycles and contains 2 index reads, in which i7 is read first, followed by the i5 sequences.