A conditional PiggyBac transposition system for genetic screening in mice: discovery of oncogenic networks in pancreatic cancer
Genomic DNA was isolated, sheared to a mean fragment length of 250bp on a Covaris AFA sonicator and ligated to a custom Splinkerette adapter. Transposon-containing fragments were enriched with 12 cycles of transposon-specific PCR for both the 5’ and 3’ transposon ends in separate libraries. Barcoding of individual samples and completion of Illumina adapter sequences was achieved with an additional 8 cycles of transposon specific PCR and a custom array of 96 unique barcoding primers. After size selection via magnetic bead purification (Beckman Ampure XP) and quantification with qPCR, libraries were pooled equimolarly in two separate 5’ and 3’ pools and sequenced on the Illumina MiSeq Desktop Sequencer (Illumina).