GSM8879294: WT_day9_iNs_AraC_chr19_amplicon_seq_controlforPKi; Homo sapiens; OTHER
For Targeted deep sequencing (amplicon-seq), collected neurons and extracted genomic DNA using DNeasy Blood & Tissue Kits (QIANGEN cat. No. 69506) following the manufacturer's protocol. For Targeted deep sequencing (amplicon-seq), amplification of targeted regions in a 25μl PCR reaction (12.5μl 2x Kapa HiFi HotStart Ready Mix, 1μg of genomic DNA, 1μM of each forward and reverse primer, and ddH2O to make 25μl) with the following PCR thermocycler program: 95°C 3min; 25 cycles of 95°C 30s, 60/64°C 15s (adjust according to Tm of primers), 72°C 30s; final extension 72°C 5min; hold at 4°C. Separated the PCR products on a 2% agarose gel and recovered the targeted DNA band by the QIAquick Gel Extraction Kit (QIANGEN, cat. No. 28704). Subsequently, Nextera index sequences were added by performing another round of PCR in 50μl reactions (25μl 2x Kapa HiFi HotStart Ready Mix, 100ng of purified DNA, 5μl of each Nextera XT index i5 and i7 primers, and ddH2O to make 50μl) with the following PCR thermocycler program: 95°C 3min; 8 cycles of 95°C 30s, 55°C 30s, 72°C 30s; final extension 72°C 5min; hold at 4°C. PCR products were separated on a 2% agarose gel and the targeted DNA band was recovered by the QIAquick Gel Extraction Kit.