Targeted Amplicon NGS-sequencing of Homo sapiens : HEK293T cells , transfected with EMX1sg7 sgRNA and hyBE4max plasmid, replicate 3, sequenced at the EMX1sg7 locus
A sequencing library with indexing primers that include unique i5 and i7 indexes, as well as P5 and P7 adapter sequences, was established via PCR. In brief, following the preparation of PCR amplicons with locus-specific primers as described above (PCR1), a second round of PCR was performed on these amplicons to barcode each sample, thereby allowing for the pooling of samples from both the same and different target sites (PCR2). The primer pairs for the second PCR contain complementary regions to the partial Illumina adapters along with the unique i5 and i7 index combinations. Individual PCR2 amplification was validated by agarose gel electrophoresis of a subsample, and  PCR2 samples were pooled, run on a 1% agarose gel, with specific bands excised and cleaned up using the Monarch DNA Gel Extraction Kit (NEB), measured by Qubit 4 Fluorometer (Thermo) using the Qubit dsDNA BR Assay Kit, and pooled to equimolarity. Samples were sequenced by GeneWiz (Azenta Life Sciences) using the Sequencing only service with a 5% PhiX spike-in on an Illumina MiSeq with 2 x 250 bp, paired-end sequencing, at a depth of approximately 10 million reads.