RNA-Seq Analysis of C. difficile Transcriptome

C. difficile strains were cultured on 70:30 sporulation agar as described above and harvested at H₁₂ into 6 ml of 1:1:2 ethanol:acetone:dH₂O solution and stored at −80°C. RNA was isolated and Dnase-I treated (Ambion). Samples were sent to Microbial Genomics Sequencing Center (MiGS; Pittsburgh, PA) where library preparation was performed using Illumina’s Stranded Total RNA prep Ligation with Ribo-Zero Plus kit and 10bp IDT for Illumina indices. Sequencing was done on a NextSeq200 giving 2×50bp reads. Demultiplexing, quality control, and adapter trimming was performed with bcl-convert (v3.9.3; Illumina; see reference above). Using Geneious Prime v2022.2.2, the reads were mapped to the reference genome (630∆erm; NC_009089.1). The expression levels were calculated and then subsequently compared using DESeq2 (80 (link)).