Tn-seq of Staphylococcus aureus LAC following intraceullar survival in primary human macrophages replicate 1
DNA was sheared in a Q880R sonicator. Cytosine tails were added with terminal deoxynucleotidyltransferase (TdT), followed by 2 PCRs. The primers olj376 (5-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGGGGGGGGGGGGGGG-3) and the transposon-specific 5-biotinylated primer PCR1-mariner-Bio (5- ACTCACTATAGGAGGGCGGGAATCATTTGAAGGTTGGTAC -3) was used for the first PCR, and an Illumina barcoded primer and the transposon-specific primer PCR2-Aa (5- AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNNCGTCTAGAGACCGGGGACTTATC -3) was used for the second PCR where the variable region (NNNNN) ranged from 3Ns to 9Ns.