RNA-Seq Analysis of C. difficile Response to UroA

Four independent biological replicates of C. difficile CD2015 were grown in BHI medium supplemented with 25 µM UroA or vehicle (DMSO) for 24 h. Following growth, cells were pelleted, supernatant was removed, 1 mL of RNA later was added, and the pellet was stored at −80°C until needed. Total RNA was extracted using the RNeasy kit (Qiagen), and residual DNA was removed using the TURBO DNA-free Kit (Invitrogen). RNA-Seq was performed at SeqCoast Genomics. RNA samples were subjected to ribosome depletion and sequenced on an Illumina NextSeq 2000 platform using a 300-cycle flow cell kit to produce 2 × 150-bp paired reads. After demultiplexing, read trimming, and FastQC analysis, transcript expression was determined using Salmon in Python 3.10 and subsequent analysis with DESeq2 and apeglm in R v4.3.0 (63 – (link)66 (link)).