title: Tn-seq - Initial library - PA14
design: Transposon mutants were scraped from solid BHI agar into 1 mL 1X Buffer A (Goodman AL, Wu M, & Gordon JI (2011) Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries. Nat Protocols 6(12):1969-1980) + 0.1% SDS and homogenized in a Mini-Beadbeater (Biospec) in 2 mL vials preloaded with Lysing Matrix B (MP Biomedicals) 3-5 times for 1 minute each, resting on ice in between each pulse. Proteinase K was then added to 1 mg/mL, and samples were incubated overnight at 50°C. Samples were then homogenized once more as above, separate sections from the same wound were pooled, and samples were extracted with an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol pH 8.0. DNA was ethanol precipitated from the aqueous phase, and was resuspended in 200-500 μL water after extensive pellet washing with 75% ethanol. Tn-seq sequencing libraries were prepared by a modified version of a published protocol (Klein BA, et al. (2012) Identification of essential genes of the periodontal pathogen Porphyromonas gingivalis. BMC Genomics 13:578). DNA was sheared to approximately 400 bp in a Q880R Sonicator (Qsonica). 2.5 μg of DNA was treated with terminal deoxynucleotidyl transferase (TdT, Promega) in a 50 μL reaction with 2.5 μL 9.5 mM dCTP / 0.5 mM ddCTP, 10 μL 5x TdT reaction buffer, and 1.25 μL rTdT at 37°C for one hour. DNA was then purified with 0.8X volume Agencourt AMPure XP beads (Beckman Coulter, Inc.) and eluted into 30 μL water. 500 ng of purified, TdT-treated DNA was used as template in a PCR reaction 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) using the following program: 95°C, 5’; 10x(94°C, 30”; 60°C, 30”; 68°C, 2’); 68°C, 10’. DNA was then purified with 0.8X volume Agencourt AMPure XP beads (Beckman Coulter, Inc.) and eluted into 50 μL water. Biotinylated PCR products were bound to Streptavidin-coupled Dynabeads (Invitrogen) as described (Goodman AL, Wu M, & Gordon JI (2011) Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries. Nat Protocols 6(12):1969-1980). The beads were then resuspended in a PCR mix containing the primers K2PTS-T8OE-2 (5’- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCGTCCAGGACGCTACTTGTG -3’) and BC41 (5’- CAAGCAGAAGACGGCATACGAGATATTATAGTGACTGGAGTTCAGACGTGTG-3’) performed with the Expand Long Template PCR System using buffer 2 (Roche) using the following program: 95°C, 5’; 15x(94°C, 30”; 58°C, 30”; 68°C, 2’); 68°C, 10’. The final library in the supernatant was removed from the beads on a magnetic stand, purified with 0.8X volume Agencourt AMPure XP beads (Beckman Coulter, Inc.), and eluted into 15 μL water. Libraries were sequenced at the Genome Sequencing and Analysis Facility at the University of Texas at Austin on a HiSeq 2000 (Illumina) on a 1x100 single end run.