GSM5964508: DMSO_2; Clostridioides difficile; RNA-Seq
After 30 min, one volume of RNAprotect bacterial reagent (Qiagen) was added followed by centrifugation (4000 x g for 10 min). Cells were resuspended in 700 µl of Qiazol lysis reagent (Qiagen) and lysed in a Fastprep cell disruptor (Qiagen) for 5 min at force 50. Total RNA was extracted using the RNeasy Mini Kit (Qiagen), according to manufacturer's instructions. Integrity of RNA was estimated on an Agilent Bioanalyzer 2100 with all samples possessing an RNA Integrity Number (RIN) >7. Ribosomal RNA was depleted with NEBNext rRNA Depletion Kit (New England Biolabs). Libraries were prepared from total RNA with the TruSeq Stranded mRNA Library Prep Kit according to the manufacturer's instructions (Illumina, PN 20020595). Libraries were analyzed for insert size distribution using the 2100 BioAnalyzer High Sensitivity kit (Agilent). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (ThermoFisher) and by low pass sequencing with a MiSeq nano kit (Illumina). Paired end 100 cycle sequencing was performed on a NovaSeq 6000 (Illumina).