GSM4982619: sequencing of CGA-2-1 gene in HeLa-iCas9-SerCGA sgRNA cells, day 4 of iCas9 induction, repeat2; Homo sapiens; OTHER
PureLink genomic DNA mini kit (Invitrogene; K182000) genomic DNA was used as template for PCR to amplify specifically the tRNA isodecoder genes of the CRISPR- targeted tRNAs in the population. PCR reaction was conducted using KAPA HiFi HotStart ReadyMixPCR (X2) (kapabiosystems; KK2601), 10µM of each primer and 100ng of genomic DNA in a 60µl total volume reaction, for 25 cycles. Primers and annealing temperatures are listed (Figure2- source data 1). After PCR validation using agarose gel, the PCR product was purified with SPRI-beads using left-side size selection protocol in which the PCR product and beads were mixed at 1:1.5 ratio. Then, each sample was barcoded with a different Illumina barcode while the Forward primer was fixed and the reverse primer included the different barcodes. The barcoding PCR reaction that was conducted using KAPA HiFi HotStart ReadyMixPCR Kit (X2), 10µM of Illumina primers and 0.35-0.7ng of PCR product in a 10µl total volume reaction, for 15 cycles. Additional clean-up step was performed using SPRI beads with left-side size selection protocol in which the PCR product and beads were mixed at 1:1.5 ratio. Samples were pooled in equal amounts and sequenced.