hns/rpoS* strain WN341 isolated from microarray analysis
Genomic DNA was prepared from ~4x10^9 mid-stationary phase bacteria using the Qiagen DNeasy kit, sheared to ~400 bp using a Covaris S2 ultrasonicator, blunted with the End-IT DNA Repair kit (Epicentre), and A-tailed using Exo-minus Klenow (Lucigen) and dATP. Custom adapter sequences with 8 nucleotide indices located 3' of each insert were ligated to the A-tailed fragments using the Fast-link ligation kit (Epicentre). Ligated DNA of approximately 500 nt of total length was isolated by 2% agarose TAE gel electrophoresis, with narrow bands excised using GeneCatcher disposable gel excision tips (the Gel Company) and purified using the Qiagen Gel purification kit. To enrich for properly ligated samples, approximately 4% of the library was amplified for 16 cycles using common primers.
University of Toronto
WN341