Preparation of cDNA library for RNA-seq

The cDNA library preparation method was carried out as previously described⁵⁷ and summarized in Supplementary Fig. S6. Briefly, total RNA was extracted from dark-grown and BL-exposed cell cultures (500 μl of culture with OD₆₀₀ = 1.0) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. After quality and quantity measurements using BioAnalyzer 2100 (Agilent, Palo Alto, CA, USA), the RNA was treated with RNase-free DNase I (Thermo Scientific, Waltham, MA, USA) at a concentration of 1 U/μg to remove residual genomic DNA. To remove the rRNA, 2.5 μg total RNA was treated with a Ribo-Zero bacterial rRNA removal kit (Illumina, San Diego, CA, USA) following the manufacturer’s instructions. The rRNA-depleted RNA pellets obtained after the ethanol precipitation step were re-suspended in 10 μl RNase free water, and then samples were fragmented using a TruSeq mRNA sample preparation kit (Illumina, USA). The cleaved short RNA fragments were used for first-strand cDNA synthesis using random hexamer primers, and then the second strand was synthesized using DNA polymerase I and RNase H. The double-stranded cDNAs were purified with AMPure XP beads (Beckman Coulter, Brea, CA, USA) and eluted with resuspension buffer followed by 3′-end adenine nucleotide addition. Finally, sequencing adaptors were ligated to the fragments and cDNA fragments were enriched by PCR amplification. Enriched cDNA libraries were used for cluster generation and sequencing. Paired-end sequencing of the cDNA libraries (dark- and BL-treated) of wild-type and knockout V. cholerae cells was performed in duplicate (biological replicates) using the Illumina MiSeq sequencing platform (Illumina).