Tn-seq - MOPS-Sputum - PAO1 - 1
Total DNA was prepared by phenol-chloroform extraction and sheared to ~500 bp by sonication. 500-1000ng of DNA was used as a template in a 99 cycle linear PCR reaction with 5' biotinylated primer 5’-ATAAGAATGCGGCCGCGGGATGGAAAACGGGAAAGGTTCCGTCCAGGACGCTACTTGTG-3’. PCR products were bound to paramagnetic streptavidin-coupled beads, a second strand was synthesized on the bead, and DNA was eluted from the beads by NotI-HF digestion. Eluted DNA was prepared for sequencing with the NEBNext DNA Library Prep Master Mix Set for Illumina (New England Biolabs).