RNA for RNA-Seq was extracted from WT, sigE−, sigK− and spoIIID−C. difficile after 18 hours of growth on 70:30 sporulation media as previously described (Fimlaid et al ., 2013 (link)). Briefly, RNA was extracted using a FastRNA Pro Blue Kit (MP Biomedical) and a FastPrep-24 automated homogenizer (MP Biomedical). Contaminating genomic DNA was depleted using three successive DNase treatments, and samples were tested for genomic DNA contamination using quantitative PCR for 16S rRNA and the sleC gene. DNase-treated RNA (5 µg) was mRNA enriched using a Ribo-Zero Magnetic Kit (Epicentre), and the quality of total RNA was validated using an Agilent 2100 Bioanalyzer.
RNA isolated for qRT-PCR was processed identically except that mRNA enrichment was done using an Ambion MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen). Reverse transcription of enriched RNA was done using the SuperScript® First Strand cDNA Synthesis Kit (Invitrogen) with random hexamer primers.
RNA isolated for qRT-PCR was processed identically except that mRNA enrichment was done using an Ambion MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen). Reverse transcription of enriched RNA was done using the SuperScript® First Strand cDNA Synthesis Kit (Invitrogen) with random hexamer primers.
