PB_GGG_20101108
About 400,000 Arg+ Ura+ colonies derived from DY1432, DY1433 and DY1434 upon transposition induction for 2 days in liquid EMM medium without thiamine were harvested and pooled. Genomic DNA was extracted from 30 OD600 units of cells and fragmented to the size of 250 ~1000 bp by sonication. The fragmented DNA in the size range of 250~750 bp was purified from 2% agarose gel with GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). A DNA adaptor composed of oligo 496 and 497 was ligated to the DNA fragments with NEBNext DNA Sample Master Mix Set 1 (New England Biolabs). To amplify the PB insertion site-flanking sequences, we performed 30 cycles of PCR with a PB specific primer close to the terminal repeats (oligo 128) and a primer annealing to the adaptor (oligo 498). After purification using the GFX kit, a further round of 30-cycle PCR was performed with primers oligo 145 and Seqr to add sequences needed for Illumina sequencing. Forty-two-cycle single-end sequencing was carried out using an Illumina Genome Analyzer II. The sequencing primer oligo 594 was designed such that the first three bases of PB specific reads were GGG from PB terminal repeats followed by PB-flanking genomic DNA sequence. (Indexed-Bioneer barcodes are also included)