GSM8764160: Clone D-2; Homo sapiens; OTHER
cDNA for amplicon sequencing was prepared using SuperScript IV reverse transcriptase. Primers designed to bind across genetic variant region in gDNA and cDNA were used to prepare amplicons for sequencing. PCR amplicon products corresponding to the SNV sites in WT and VUS MED12 were prepared from extracted gDNA with forward and reverse amplicon primers and Phusion High-Fidelity PCR Master Mix with HF Buffer (Thermo Scientific). Purified samples were barcoded with TruSeq primers (IDT) and MED12 sequences determined with paired-end, 250bp DNA amplicon sequencing with a MiniSeq Mid-Output Kit, 300 cycles.