GSM7978742: Δhns KDS1 rep1 - RNA-seq; Vibrio cholerae; RNA-Seq
RNA isolation. Bacterial strains of interest were grown to an OD600 of 0.3 in 4 mL culture tubes and mixed 1:1 with ice-cold methanol in a 15 mL falcon tube, under culture conditions equivalent to the IPOD experiments described above. Methanol-treated samples were pelleted at 7,000 x g at 4C, and the resulting pellets were washed in ice-cold phosphate-buffered saline. The washed pellets were resuspended in 200 µL TRI Reagent (Millipore/Sigma) and incubated for 5 minutes at room temperature. The samples were mixed with 40 µL chloroform, vortexed, and incubated for 10 minutes at room temperature. The chloroform-treated samples were then centrifuged at 12,000 x g for 10 min at 4C. Following centrifugation, the upper (aqueous) phase was collected, promptly mixed with 110 µL 2-propanol and 11 µL pH 6.2 3M sodium acetate, and mixed vigorously. The samples were centrifuged at 12,000 x g for 15 min at 4C, and the pellets were washed twice with 500 µL of 75% ethanol. The washed pellets were incubated uncapped at 65C for 3 minutes to evaporate any residual ethanol and resuspended in 20 µL of diethyl dicarbonate (DEPC) treated water. rRNA depletion and RNA-sequencing library preparation. Purified RNA was subjected to Baseline-ZERO™ DNase digestion in a 100 μL reaction of 20 μL of purified RNA, 5 μL Baseline-ZERO™ DNAse enzyme (LGC Biosearch Technologies, Pt# E0110-D1, 1 U/μL), 10 μL 10X Baseline-ZERO™ DNase Reaction Buffer, 2 μL RNase Inhibitor, Murine (NEB, #M0314S/L) and nuclease-free water at 37C for 30 minutes. The RNA was purified with Zymo RNA clean and concentrate kit and eluted with nuclease-free water. The quality of purified RNA was assessed on a 2% agarose gel with guanidine thiocyanate and quantified with a NanoDrop spectrophotometer. Purified RNA was depleted of ribosomal RNA (rRNA) using a NEBNext® rRNA Depletion Kit (Bacteria) (NEB, #E7850L/X) and library preparation was done using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, #E7760S/L), according to the manufacturer's instructions. The libraries were assessed for quality with agarose gel electrophoresis and quantified with a Promega Quantifluor High Sensitivity dsDNA kit.