Illumina HiSeq 2000 paired end sequencing
Vibrio cholerae co969 smooth colonies grown at 22ºC during 24 h
Total RNA from V. cholerae co969 smooth colonies grown at 22ºC during 24 h was isolated as follows: 8 colonies grown under the desired conditions were collected by scraping, pooled together in an Eppendorf tube for RNA extraction and stored at -20ºC. Pellets were resuspended in 400 µl of solution containing 10% glucose, 12.5 mM Tris pH 7.6 and 5 mM EDTA and, after adding 60 µl 0.5 M EDTA, cells were disrupted on a bead beater (4ºC, maximum speed for 1 min 15 sec) in the presence of 0.5 ml of acid phenol. After centrifugation, 1 ml Trizol (Ambion) was added to the supernatant, incubated at room temperature for 10 min, 100 µl chloroform:IAA were added, mixed by vortex for 10 sec, and centrifuged at 14.000 rpm 4ºC for 15 min. After two more chloroform:IAA extractions, the RNA was precipitated by mixing the aqueous phase with 0.7 volumes isopropanol, incubated for 30 min at -20ºC, centrifuged at 14.000 rpm 4ºC for 30 min, and washed with 70 % ethanol. RNA pellets were then resuspended in 200 µl H2O and subjected to two consecutive DNaseI (Roche) treatments for 1h at 37ºC, in the presence of RNase inhibitor. After a phenol:chloroform:IAA and a chloroform:IAA extraction, the RNA was precipitated by mixing the aqueous phase with 3 volumes of 95% ethanol and 1/10 volumes of 3M sodium acetate pH 4.6, incubated for 30 min at -20ºC, centrifuged at 14.000 rpm 4ºC for 30 min, washed with 70 % ethanol, and finally resuspended in 100 µl H2O. The integrity of the RNA was checked on a 1.2% agarose gel and with RNA6000 Nano Assay using the Agilent 2100 Bioanalyzer (Agilent Technologies) and RNA samples were quantified using a Qubit 2.0 Fluorometer (Life Technologies). To enrich mRNA, ribosomal RNA (rRNA) was removed from the samples using the Ribo-Zero rRNA removal kit (Illumina). cDNA preparation and sequencing reactions were conducted in the GeneCore Facility of the European Laboratory for Molecular Biology (EMBL, Heidelberg). Construction of mRNAseq libraries was conducted using the TruSeq Stranded Total RNA Library Prep Kit (Illumina) following the manufacturer's recommendations.