GSM2701508: 4_Pang_Tn_aTP315_2; Staphylococcus aureus subsp. aureus NCTC 8325; Tn-Seq
Genomic DNA was extracted. Transposon-sequencing (Tn-seq) was adapted from Zhang et al (PMID: 23028335). Briefly, genomic DNA isolated from each library was sonicated to 200-400 bp fragments. Nicked ends were repaired (Quick Blunt kit, NEB), and dA-tails were added with Taq polymerase (NEB), followed by ligation of adapters using T4 ligase (NEB). Transposon-junctions were PCR-amplified using primers binding to adapter and transposon individually. PCR products were purified and incubated with the endonuclease NotI (NEB) to remove any contaminating DNA derived from the transposon plasmid. Finally, a second round of PCR was performed on 100 ng of each sample, using primers that contain barcode and illumina P5/P7 and sequencing primers. A 200-400 bp product was gel-purified.