GSM7769253: GCPS11484; Homo sapiens; OTHER
For this assay, genomic DNA was harvested from cells using a Quick-DNA 96 Plus Kit (Zymo Research). The DNA concentration was measured using a Qubit 1X dsDNA BR assay kit (ThermoFisher). Purified genomic DNA was then used to amplify the HBB locus via polymerase chain reaction (PCR). The PCR products were diluted using nuclease-free water to serve as the template DNA for targeted NGS library prep. An Aglient Tapestation was used to confirm the PCR product for each sample was the expected size (1410bp). A second PCR with primers carrying partial Illumina adapters was performed to amplify a 142 base pair sequence that includes the region of the HBB locus that is to be corrected during the genome correction process. The PCR products were diluted again to serve as templates in a third PCR reaction using Nextera XT index primers. This third PCR reaction was used to assign unique identifiers to each sample and to add the full length adapter sequences necessary for Illumina sequencing. The size of the PCR products was assessed on an Agilent BioAnalyzer. PCR products were then pooled, purified using a Qiagen PCR purification kit, and quantified using PicoGreen in order to ready the PCR products for sequencing. Quick-DNA 96 Plus Kit (Zymo Research).