Targeted Resequencing of gene editing human hematopoietic stem and progenitor cells
DNA library for HTS was prepared as described previously (Hoban et al., 2015; Lomova et al., 2019). Briefly, an outer PCR was performed on genomic DNA to amplify a 1.1kb region of interest (using Outer PCR Forward (Fwd) and Reverse (Rev) primers). A second PCR was performed to add a unique index to the PCR product of each sample to be sequenced (read1/read2 and P5/P7 primers). The PCR products with the indexes were mixed at equal concentrations, which was determined by densitometry of the PCR products and analyzed by gel electrophoresis, to create a pooled library. The pooled library was purified twice using AMPure XP beads (Beckman Coulter Inc.; Brea, CA) and then quantified using ddPCR (QX 200; Bio-Rad Laboratories Inc.; Hercules, CA). HTS was performed at UCLA Technology Center for Genomics & Bioinformatics (TCGB) using MiSeq 2x150 paired-end reads (Illumina Inc; San Diego, CA).