GSM8353690: split ePE3 with untethered MMLV-RTΔRH at EMX1 loci - Rep1 -I7; Homo sapiens; OTHER
Genomic DNA (gDNA) was isolated using TIANamp Genomic DNA Kit (TIANGEN #DP304) according to the manufacturer's protocol. The region that integrated with genomic DNA including RT template was PCR amplified from genomic DNA for HTS. For the first round of PCR, genomic sites of interest were amplified from 150 ng of genomic DNA using Q5 PCR Master Mix (New England Biolabs #M0544S) with the primers containing Illumina forward and reverse adapters. 50 uL of PCR 1 reaction was performed with forward and reverse primers each at 0.25 uM, ~1 uL of genomic DNA extract, and 25 uL of Q5 PCR Master Mix. To add a unique sequencing index, 50 uL of a given PCR 2 contained 2.5 uL of previous step unpurified PCR product; a unique forward or reverse primer pair, each at 0.25 uM; and 25 uL of Q5 PCR Master Mix. Primers are listed in Table S1. PCR 2 products were purified by DNA AMPure XP beads (Beckman Coulter #A63882) and eluted with 25 uL of water. DNA concentration was quantified by Nanodrop One (Thermo Fisher Scientific).