GSM2334638: MT02_128-Rep_1; Staphylococcus aureus; RNA-Seq
RNA was isolated with an RNeasy Mini kit (QIAGEN) according to the instructions of the manufacturer. To remove the DNA template, RNA was treated with RNase-free DNase I (New England, Biolabs® Inc, Frankfurt am Main, Germany). Total RNA of MT02-resistant and wild-type strain JE2 was extracted with the RNeasy (Qiagen) or the MirVana isolation kit (Thermo Fisher Scientific). Fragmentation of messenger-enriched RNA was performed using zinc. Specific tags were ligated before reverse transcription to conserve strand orientation. Single-stranded ligation of the 3′ Illumina adapter was followed by single-stranded ligation of 5′ bar-coded adapter. Reverse transcription and PCR amplification were performed to generate a cDNA colony template library, and gel purification with selection of fragments of 20 to 300 nt was performed.  For HTS experiments, total RNA was treated with the MICROBExpress kit (Thermo Fisher Scientific) to limit contamination by multicopy structural rRNAs, following manufacturer's instructions with slight modifications. The maximal amount of total RNA per tube was limited to 4–5 µg because previous experiments showed important contamination levels when using 10 µg. Between each purification step, RNA quality and quantity were determined by Bioanalyzer (Agilent) using the RNA Nano chips, and quantified using the ND-8000 (Thermo Fisher Scientific).
GEO Accession
GSM2334638