Transcriptional Profiling of T6SS Vibrio cholerae

Overnight cultures of T6SS+, T6SS−, and WT Vibrio cholerae were grown at 37°C in lysogeny broth (LB) (n = 2 biological replicates). RNA was extracted from these cultures using an Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). An RNA library was prepared using the NEB Ultra II directional RNA library prep kit (New England Biolabs, Ipswitch, MA, USA) and the QIASeq Fast select ‐RNA HMR kit (Qiagen, Hilden, Germany) was used for bacterial rRNA depletion. The completed bacterial RNA library was sequenced on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). To analyze the transcriptomic data, we first trimmed adapters and filtered low‐quality reads using Trimmonatic (v0.39) (Bolger et al., 2014 (link)). We then perform alignment of paired reads to reference contigs of V. cholerae C6706 (NCBI RefSeq assembly GCF_009763945.1) using STAR (v 2.7.11a) (Dobin et al., 2013 (link)) to create binary alignment files (BAM) sorted by genomic coordinates. We counted aligned fragments to all annotated loci in NCBI Refseq annotation using featureCounts (v2.0.6) (Liao et al., 2014 (link)). Fragment counts were filtered for low expression and used for differential expression analysis using DESeq2 in R (Love et al., 2014 (link)).