Tn-Seq of cdnL Caulobacter crescentus replicate 2
Cells were grown in a large culture (1 liter) in PYE medium to mid-log (0.4-0.6), washed of excess Mg2+ with 10% glycerol, and mutagenized with the Ez-Tn5 <Kan-2> transposome (Epicentre). Cells recovered by shaking at 30 C for 90 minutes, then plated on kanamycin containing plates for 3 days at 30 C in order to yield roughly 100-500 colonies per plate. Libraries were grown at 30 C and comprised ~100,000-200,000 colonies each. Mutants were pooled into one library by scraping colonies from the surface of the agar and added into ~25-40 mL PYE. Pooled libraries were shaken to yield a homogenous slurry and sterile glycerol was added to 20%. Libraries were then frozen in liquid nitrogen and stored at -80 C. Two libraries of each genetic background were prepared individually and compared as biological replicates. Genomic DNA was extracted from one aliquot of each library using DNeasy Blood and Tissue Kit (Qiagen). Tn-Seq libraries were prepared for Illumina Next-Generation sequencing through sequential PCR amplifications using arbitrary hexamer primers and Tn5-specific primer facing outward for the first round, and indexing primer sets that include unique molecular identifier to filter artifacts arising from PCR duplicates for the second round. Libraries were then pooled and sequenced using Illumina primers at the University of Massachusetts Amherst Genomics Core Facility on the NextSeq 550 (Illumina). Reads were aligned to the Caulobacter vibrioides NA1000 reference assembly (accession #CP001340)