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Fig. 1 | BMC Genomics

Fig. 1

From: A simplified transposon mutagenesis method to perform phenotypic forward genetic screens in cultured cells

Fig. 1

Overview of cell-based Sleeping Beauty (SB) mutagenesis screens. (1) Cells are engineered to express the SB100X transposase. This is done using a piggyBac transposon expression system using the hyPBase transposase to stably deliver the SB100X transgene. (2) Once SB100X-expressing cells are selected, cells are transfected with the T2-Onc3 plasmid, or similar mutagenic SB transposon vector, and cells with the desired phenotype are selected from among the population of mutagenized cells. (3) Genomic DNA is collected from each population of selected and control cells, and each DNA sample is subjected to a ligation-mediated PCR approach to isolate the transposon-genome junctions. Individual LM-PCR libraries are then multiplexed and sequenced on the Illumina platform. (4) Raw sequences are trimmed, mapped to the reference genome, and filtered based on user-defined criteria. (5) Finally, filtered sequences are combined and analyzed to identify candidate genes enriched for transposon insertions in selected but not control cells

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