GSM7156345: WT replicate 2; Brucella ovis; RNA-Seq
Cells were incubated on rotor at 37˚C in 5% CO2 for 7h. 9 ml of each culture were spun down and the pellets were resuspended in 1 ml TRIzol. Samples were stored at -80˚C until RNA extraction. To extract the RNA, samples were thawed and incubated at 65˚C for 10 minutes. 200 µl of chloroform was added to each sample and mixed by vortexing for 15 seconds. Samples were then incubated at room temperature for 5 minutes. Samples were spun at 17,000 x g for 15 minutes at 4˚C and the aqueous layer was collected. Sample was mixed with 500 µl isopropanol and stored at -20˚C for 2h. After thawing, samples were spun down at 13, 300 x g for 30 minutes at 4˚C. Supernatants were discarded and 1 ml of ice cold 70% ethanol was added. Samples were spun down at 13,300 x g at 4˚C for 5 minutes. Supernatants were discarded and pellets were let completely dry. 100 µl RNAse-free H2O was added to each sample and incubated at 60˚C for 10 minutes. RNA samples were cleaned up using RNeasy Mini kit (Qiagen). RNA libraries for RNA-seq were prepared using Illumina Stranded RNA library preparation with RiboZero Plus rRNA depletion.