Transposon-chromosome junctions were prepared and amplified for high-throughput sequencing following protocols described by Fu et al. (13 (link)).
Genomic DNA was extracted from 0.5 ml aliquots of each Tn library (8 in total: 4 intact- and 4 HI-NHS samples), sheared into ~500-bp fragments by ultrasonication using a Covaris M220 system, end-repaired (Quick Blunting Kit; NEB) and A-tailed by Taq polymerase (NEB). Adaptor DNA sequences (Adapter M1.0 and Adapter M2.0; Supplemental Table III) were then ligated to the fragmented DNA and PCR reactions carried out to amplify DNA fragments containing the Tn-chromosomal junction sequences along with the appropriate sequences required for Illumina sequencing (such as P5 and P7 hybridization sequences and barcodes) using primers Tn5-PCR F and Multiplex index 1–12, depending on the number of samples pooled for sequencing (Supplemental Table III). The final PCR products were purified on a 2% agarose gel and DNA fragments of 200–400-bp in length isolated. Equimolar DNA fragments from each library were combined and sequenced using the MiSeq Reagent Kit v2 50 cycles (Illumina) along with the custom sequencing primer Tn5-Seq (Supplemental Table III).