Tn-seq - Polymyxin B - replicate 2
Samples were extracted with phenol: chloroform:isoamyl alcohol, pH 8.0. DNA was ethanol-precipitated. DNA was sheared to ∼300 bp in a Q880R Sonicator (Qsonica). The sheared DNA was TdT-treated. TdT-treated DNA was used as template in a PCR using the primer olj376 (5’-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGGGGGGGGGGGGG-3’) and the 5’-biotinylated primer K2PTS-T8OE-1-bio (5’-GGGTTTTCCCAGTCACGACGTTG-3’) and performed with the Expand Long Template PCR System using buffer 2 (Roche). DNA was then purified with Agencourt AMPure XP beads (Beckman Coulter, Inc.) Biotinylated PCR products were bound to Streptavidin-coupled Dynabeads (Invitrogen). The beads were then resuspended in a PCR mix containing the primers K2PTS-T8OE-2 (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCGTCCAGGACGCTACTTGTG-3’) and primer containing an illumina TruSeq barcode BC__ (5’-CAAGCAGAAGACGGCATACGAGATxxxxxxGTGACTGGAGTTCAGACGTGTG-3’) performed with the Expand Long Template PCR System (Roche). The final library in the supernatant was removed from the beads on a magnetic stand, purified with Agencourt AMPure XP beads (Beckman Coulter, Inc.).