title: GSM4830178: PTEN_VAMP-seq_Exp1 (Multidimensional PTEN missense variant analysis(; Homo sapiens; OTHER
library_construction_protocol: Cells were transferred into a microfuge tube, pelleted and stored at -20˚C. Genomic DNA was prepared using the Dneasy kit (Qiagen). Eight 50 μL first-round PCR reactions were each prepared with a final concentration of ~50 ng/μL input genomic DNA, 1x Kapa HiFi ReadyMix, and 0.25 μM of the KAM499/JJS_501a primers. The reaction conditions were 95 °C for 5 minutes, 98 °C for 20 seconds, 60 °C for 15 seconds, 72 °C for 90 seconds, repeat 7 times, 72 °C for 2 minutes, 4 °C hold. Eight 50 μL reactions were combined, bound to AMPure XP (Beckman Coulter), cleaned, and eluted with 40 μL water. 40% of the eluted volume was mixed with 2x Kapa Robust ReadyMix; JJS_seq_F and one of the indexed reverse primers, JJS_seq_R1a through JJS_seq_R12a were added at 0.25 μM each. Reaction conditions for the second round PCR were 95 °C for 3 minutes, 95 °C for 15 seconds, 60 °C for 15 seconds, 72 °C for 30 seconds, repeat 14 times, 72 °C for 1 minutes, 4 °C hold.  Amplicons were extracted after separation on a 1.5% TBE/agarose gel using a Quantum Prep Freeze 'N Squeeze DNA Gel Extraction Kit (Bio-Rad).