Instrument: NextSeq 500
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: Cells were transferred into a microfuge tube, pelleted and stored at -20˚C. Genomic DNA was prepared using the GentraPrep kit (Qiagen) or Dneasy kit (Qiagen). For TPMT: for each bin, all the purified DNA was spread over eight 25 uL PCR reactions containing Kapa Robust, primers GPS-landing-f (in the genome) and BC-GPS-P7-i#-UMI (3' of the barcode) to tag the barcodes with a unique molecular index (UMI) and add a sample index. UMI-tagging PCR were performed using the following conditions: initial denaturation 95 ˚C 2 minutes, followed by three cycles of (95 ˚C 15 seconds, 60 ˚C 20 seconds, 72 ˚C 3 minutes). The eight PCR reactions were pooled and the PCR amplicon was purified using 1x Ampure XP (Beckman Coulter). To shorten the amplicon and add the p5 and p7 Illumina cluster-generating sequences, the UMI-tagged barcodes were then amplified with primers BC-TPMT-P5-v2 and Illumina p7. This PCR was performed with Kapa Robust and SYBR green II on a Bio-Rad mini-opticon qPCR machine, reactions were monitored and removed before saturation of the SYBR green II signal, at around 25 cycles. The amplicons were pooled and gel purified. For PTEN: . 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).