title: Tn-himar-seq data used to map insertion sites
design: Tn insertions sites were mapped following the approach outlined by Wetmore et al. [doi: 10.1128/mBio.00306-15] with modifications to the PCR enrichment.  Briefly genomic DNA was extracted using guanidium thiocyanate. Four micrograms of genomic DNA in 130 ul volume were sheared using a Covaris M220 ultrasonicator using manufacturers settings to generate ~300 bp fragments.  Using the NEBNext Ultra II Library Prep Kit (New England Biolabs, E7103S) and following the manufacturers protocol, 1 ug of sheared DNA was end-repaired and A-tailed then ligated to a custom Y adapter, prepared by annealing Mod2_TruSeq and Mod2_TS_Univ.  The final product was cleaned with SPRIselect beads using a two-sided (0.6-0.25X) selection and eluted from the beads in 25 ul 10 mM Tris, pH 8.5.  The fragments containing Himar transposons were enriched with a two-step, nested PCR strategy using Q5 DNA polymerase with GC enhancer (New England Biolabs, M0491).  Primer sequences were modified from those originally described by Wetmore et al. [62].  In the first reaction the forward primer contained the TruSeq Read 1 region, a random hexamer to facilitate clustering, and a transposon-specific sequence, and the reverse primer corresponded to the TruSeq Read 2 sequence.  Compared to Nspacer_barseq_universal [62], the transposon-specific portion of the primer was extended to improve specificity in the amplification.  The second PCR reaction resulted in the addition of P5 and P7 sequences to the products as well as a 6-bp index on the P7 end.  Reaction 1 contained 0.5 uM of each of the TS_bs_T7-35 and TS_R primers, 0.2 mM dNTP, 1X Q5 reaction buffer, 1X GC enhancer, 2 Units Q5 polymerase and 10 ul of adapter-ligated fragments in a 100 ul reaction volume.  Cycling parameters were 98C for 3 min, 20 X (98C 30 s, 66C 20 s, 72C 20 s), 72C for 5 min, 4C hold.  The first reaction was cleaned with 0.9X AMPure XP beads (Beckman Coulter, A63880) and eluted in 30 ul of 10 mM Tris, pH 8.5.  Reaction 2 contained 0.5 uM of each of the primers, P5_TS_F and P7_MOD_TS_index6, 0.2 mM dNTP, 1X Q5 reaction buffer, 1X GC enhancer, 2 Units Q5 polymerase and 15 ul of cleaned reaction 1 in a 100 ul reaction volume.  Cycling parameters were 98C for 3 min, 15 X (98C 30 s, 69C 20 s, 72C 20 s), 72C for 5 min, 4C hold.  The final product was cleaned with SPRIselect beads using a two-sided (0.9-0.5X) selection and eluted from the beads in 40 ul 10 mM Tris, pH 8.5.  The amplified fragments were sequenced using a 150-cycle MiniSeq High Output Reagent Kit (Illumina, FC-420-1002) on an Illumina MiniSeq.  To aid in clustering, sequencing runs were supplemented with 20-30% phiX DNA.