VCP Gene Mutation Identification Protocol

iPSC DNA (~310 bp) was amplified by PCR (Platinum SuperFi PCR Master Mix, Invitrogen #12358) using custom Reverse primers (IDT). Primers (VCP-Ex5-F 5′-TGGAGTTGGGGAGAGGTAGGG-3′, and VCP-Ex5-R 5′-AAAATCGGATACTGGAATCAGGGAGA-3′). PCR product was digested with Sph1 HF (New England Biolabs #R3182L), and clones positive for the Sph1 digestion were purified using agarose gel (Qiagen, #28704). Before sequencing, amplicons were treated with Exonuclease I (10 U Thermo Fisher Scientific # EN0581) and FastAP™ Thermosensitive AP (1 U, Thermo Fisher Scientific #EF0654). The samples were mixed and incubated at 37°C for 15 min, followed by incubation at 85°C for 15 min. Samples were sent for sequencing and analyzed using the software FinchTV (Geospiza, Inc., WA, United States) (23 (link)).