GSM2089706: V2A.t24; Enterococcus faecalis; RNA-Seq
Cells from 30 ml culture were harvested by centrifugation for 1 min at 10 000 rpm and 4°C in a Sorvall RC6+ centrifuge (Thermo Scientific GmbH, Langenselbold, Germany). Cell pellets were immediately frozen in liquid nitrogen and stored at -80°C or directly used for RNA isolation. RNA was isolated using the ZR Fungal/Bacterial RNA MiniPrepTM Kit (Zymo Research, Freiburg, Germany) following the manufacturer's instructions. To recover total RNA including small RNAs, 1.5 volumes of absolute ethanol were added in step 5. Finally, total RNA was eluted with 50 µl DNase-/RNase-free water and stored at -80°C. RNA quantity and quality were assessed with Agilent RNA 6000 Nano and Pico Kits using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Residual contaminating DNA was digested with DNA-freeTM DNase (Ambion, Carlsbad, USA). To remove rRNAs and tRNAs from the total RNA, MICROBExpressTM and MEGAclearTM Kits were applied. After purification, 500 ng of the enriched (m)RNA was fragmented with RNase III. Fragmented (m)RNA (50-100 ng) was used for the preparation of barcoded whole transcriptome cDNA libraries using the Ion Total RNA-Seq Kit v2 and Ion XpressTM RNA-Seq Barcode 1-16 Kits. Finally, strand-specific, multiplexed RNA sequencing was carried out on Ion PITM chips in an Ion ProtonTM Sequencer. All kits and devices for RNA sequencing were from Ambion, Carlsbad, USA and used according to the manufacturer's instructions.
GEO Accession
GSM2089706