70% ethanol, resuspended in 30–50 μL TE buffer with 0.1 mg/mL RNase A (Thermo Fisher Scientific), and allowed to dissolve at room temperature overnight. hPSC clones were screened by PCR amplification of a small region surrounding the targeted site using BioReady rTaq DNA Polymerase (Bulldog Bio) and the following cycling conditions: 94°C for 5 min, [94°C for 30 sec, 54–56.5°C for 30 sec, 72°C for 30 sec] × 40 cycles, 72°C for 5 min. The following primer pairs were used: for rs2277862, F: 5′-TGCTGGACCCACACTTCATA-3′ and R: 5′-CTCAGTCCCTCTCCCTCCTT-3′; for rs10889356, F: 5′-CCATTAGGTCACTTGCCAGA-3′ and R: 5′-ACAGGGGGATTCTGTCTAAAA-3′; for rs10872142, F: 5′-GCTGATCTTAGCTGGGCTTG-3′ and R: 5′-TTTGTGCTTCCTTGCATCTG-3′. PCR amplicons were separated on a high-percentage agarose gel, and clones with indels were identified based on size shifts relative to the wild-type band. Clones were confirmed by Sanger sequencing of the PCR products. Multiple mutant clones were retrieved from the frozen stock, or if possible, from the second working stock and expanded for experiments. Additionally, several clones that underwent the targeting procedure but remained genetically wild-type at the intended site were expanded as controls.
