GSM3133291: S4_LI_Cp13; Corynebacterium pseudotuberculosis; RNA-Seq
Following 6:30 hours of incubation, each culture (LI and Ct) was centrifuged for 10 minutes at 5.000 rpm (4˚ C) in 50 mL conical tubes, and the supernatants were discarded. The bacterial pellets were kept on ice and resuspended in 300 µL of Tris/EDTA/SDS (Tris HCl pH8 50 mM/L–EDTA pH8 10 mM/L) lysis buffer per 10 mL of used initial culture volume. Cell suspensions were homogenized with 0,1% of SDS and 1 mL of TRIzol® Reagent (Invitrogen™) was added (1 volume per 10 mL of used initial culture). Pellets from each tube were divided into 2 new tubes filled with 1mm diameter glass microbeads (Bertin Thechnologies). The cells were mechanically lysed using Prescellys®24, set for 2 cycles (15 seconds per cycle) at 6200 rpm with 5 seconds interval between each cycle. The tubes were immediately placed on ice following the lysis. The cooled samples were centrifuged for 10 seconds at 14.000 rpm and the supernatants were carefully transferred to a new tube for total RNA extraction. Subsequent extraction steps were carried out following TRIzol® Reagent manufacturer's recommended protocol and total RNA was eluted in 50ul of RNase free water. Traces of contaminating DNA were eliminated with the Ambion® TURBO DNA-free™ DNase kit (Invitrogen), following manufacturer's protocol. All RNA samples were subjected to ribosomal RNA depletion and purification using the RiboMinus™ Bacteria Transcriptome Isolation Kit with the RiboMinus™ and the Concentration Module (Invitrogen), as described in the manual. 5 µg of purified depleted RNA samples were used to prepare 14 individual single-end RNA-seq libraries with the Ion Total RNA-seq v2 for Whole Transcriptome Library kit (Thermo Scientific).