Comparison of relative gene expression between Vibrio cholerae co969 colonies incubated at 37°C (rugose, 37R) and 22°C (smooth, 22 Sb) by mRNA-seq. Colonies had a similar number of colony-forming units per colony, as described under experimental procedures. (A) Relative expression of vpsL and bap1 between colonies grown at 37°C vs. 22°C for co969 and co969:hapRc strains, determined by quantitative real-time polymerase chain reaction (qRT-PCR). Results are the average of three biological replicates, each replicate containing eight colonies. Error bars, standard deviation. Expression of hfq was used as a control. (B) Upper panel: MA-plot representing the log2FC against mean expression for differentially expressed genes between 37R vs. 22 Sb colonies. Black dots, significantly differentially expressed genes; gray dots, not significantly differentially expressed genes; red dots, genes encoding biofilm structural components (vpsI and vpsII clusters, rbmA, rbmC, and bap1). Lower panel: number of differentially expressed genes (up- and downregulated) between 37R vs. 22 Sb colonies, grouped by Clusters of Orthologous Groups of proteins (COGs) categories. (C) Heat map showing the differential relative expression (fold change) between 37R and 22 Sb of genes belonging to the vpsI, vpsII, and rbm clusters and bap1, encoding the Vibrio polysaccharide (VPS) and biofilm matrix proteins (RbmA, RbmC, and Bap1), respectively, and main biofilm regulators VpsT, VpsR, and HapR. (D) Validation of differential gene expression obtained by mRNA-seq by qRT-PCR. The graph represents a comparison of the relative gene expression in both the mRNA-seq analysis and qRT-PCR experiments of biofilm activators (vpsR and vpsT), genes encoding VPS (vpsL, vpsA, and vpsU) and matrix proteins (rbmA, rbmC, and bap1) between 37R and 22 Sb colonies. Values are the average of three independent qRT-PCR experiments containing three biological replicates each, each replicate containing eight colonies pooled together and three technical triplicates of each biological replicate. Error bars, standard deviation. Expression of gyrA was used as a control.