GSM8705509: edited_long_rep_2; Staphylococcus aureus; RNA-Seq
Total RNA was extracted using TRIzol (Invitrogen) according to the manufacturer's protocol after sonication with the LABSONIC M sonicator (Sartorius, Göttingen, Germany). The samples were treated with DNase I and the quality of the RNA was visualized using agarose gel electrophoresis. Ribosomal RNA (rRNA) was depleted using the RiboMinusTM Bacteria 2.0 Transcriptome Isolation Kit (Thermo Fisher Scientific). cDNA libraries were prepared from 100 ng rRNA-depleted RNA using the Ion Total RNA-Seq v2 Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, RNAs were digested with RNase III to produce 200 nt RNA fragments, which were successively treated for adapter ligation, reverse transcription, and 14 cycles of PCR amplification using the Ion Xpress™ RNA-Seq Barcode 1-16 Kit (Thermo Fisher Scientific). The quantity of the libraries was measured with the Qubit 1x dsDNA HS Assay Kit (Thermo Fisher Scientific) and the size distribution was assessed using the Agilent High Sensitivity DNA Kit on the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Template preparation and the Ion chip 540™ loading were performed on the Ion Chef system using the Ion 540™ Chef kit. The single-end sequencing was carried out on an Ion GeneStudio S5 sequencer. RNA sequencing analysis was performed in biological duplicates. For the sequencing of small RNAs, total RNA was run onto a 12% UREA-PAGE and the <150 nt RNA fraction was gel extracted . The library preparation procedure was the same as in the case of the total transcriptome analysis, except for the steps of ribosomal RNA depletion and RNase III treatment.