TagTn sequencing of IS element with TnpA, Lane 3
Plasmids with an R6K ori, a CmR marker, and inverted IStron ends (pDonorCI) were cloned in pir+ strain (sSL0281). E. coli str. K-12 substr. MG1655 transformed with a TnpA expression plasmid driven by pLac was grown in liquid LB with IPTG (0.5 mM) and cells were made electrocompetent at OD600 = 0.6. These cells were then electroporated with various pDonorCI variants, recovered for 7 hours, plated on LB Agar plates with chloramphenicol (25 gml1), and grown for ~24 hours. Surviving colonies were pooled and genomic DNA was extracted and quantified via Qubit. Approximately 100 ng of gDNA was tagmented with TnY, pre-loaded with Read 2 Nextera oligos (Supplementary Table 4). Two rounds of PCR were performed as described for targeted detection of IS excision events with oligos that annealed to either the left or right IStron end (Supplementary Table 4). Paired-end, 7676 cycle sequencing was performed on a NextSeq platform. Using BBDuk [REF], reads were then filtered for containing the proper IStron end sequence, and the flanking genomic sequence was extracted. Reads that contained the parental pDonorCI sequence were removed during this process. Flanking genomic sequences were then aligned to the E. coli genome using Bowtie2. WebLogo representations were generated using the input sequence on both the left and right IStron end, as well as the mapped genomic insertion sites.