Amplicon sequencing of HEK293T gDNA
Genomic DNA was amplified by qPCR using Phusion Hot Start II DNA polymerase with use of SYBR gold for quantification. 3% DMSO was added to all gDNA PCR reactions. To minimize PCR bias, reactions were stopped during the exponential amplification phase. 1 uL of the unpurified gDNA PCR was used as a template for subsequent barcoding PCR (8 cycles, annealing temperature 61 C). Pooled barcoding PCR products were gel-extracted (Min-elute columns, Qiagen) and quantified by qPCR (KAPA KK4824) or Qubit dsDNA HS assay kit (Thermo Fisher). Sequencing of pooled amplicons was performed using an Illumina MiSeq
SUB5098956
BE3_EMX1_Rep1