GSM8062153: D09_spegJY046_CXCR4_5GtoC_P191A_6U_rU_donor6_PE7_317; Homo sapiens; OTHER
Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen 69506) per manufacturer's instructions with an elution volume of 100 µL To assess editing efficiency, PCR was performed with 25 uL of eluted genomic DNA per sample in an 100 µL PCR reaction with KAPA HiFi HotStart ReadyMix (Roche 9420398001) with the following cycling conditions: 95 C for 3 min, 28 cycles of [98 C for 20 s, 63 C for 15 s, and 72 C for 60 s], followed by 72 C for 2 min. PCR products were purified by SPRI selection (Beckman Coulter B23317) and 2 µL eluted product was used for 8 cycles of additional PCR with KAPA HiFi HotStart ReadyMix to add Illumina sequencing adapters and indices. The final PCR products were purified by SPRI selection, quantified with Qubit 1X dsDNA High Sensitivity (HS) assay kit (Invitrogen Q33230), equimolarly pooled, and sequenced with the MiSeq Reagent Kit v2 300 cycles (Illumina MS-102-2002) with 300 cycles for R1 read, 8 cycles i7 index read and 8 cycles i5 index read.