Transcriptome Analysis of V. cholerae

Total RNA from V. cholerae strains JB58 and JB485 grown under AKI conditions for 3h were isolated using TRIzol per the manufacturer’s directions (Invitrogen) and further purified using an RNeasy kit with in column DNase treatment (Qiagen). The resulting RNA samples were assessed using a Qubit 2.0 fluorimeter (Thermo Scientific) and Agilent Tapestation 2200 (Agilent Technologies). Sequencing libraries were generated using the Illumina TruSeq RNA Access library prep kit (Illumina). Cluster generation and 75-bp single-read single-indexed sequencing was performed on Illumina NextSeq 500 (Illumina). The resulting raw reads were trimmed to remove adaptor/primer sequences. CLC Genomics Workbench version 10.1 (Qiagen) was then used to map the reads from three independent experiments to the N16961 genome [42 (link)]. The identification of differentially expressed genes was accomplished using the CLC Genomics Workbench RNA-Seq Analysis tool. Genes showing a 2-fold or greater difference in expression and a P-value and False Discovery Rate P-value of less than or equal to 0.05 were identified as differentially expressed genes. The RNA sequencing data was deposited in the National Center for Biotechnology Information Sequence Read Archive under accession number SRP109296.