Isolation of Primary RNA for Differential RNA Sequencing in Fusobacterium nucleatum

Understanding gene expression in Fusobacterium nucleatum and related species is essential for uncovering the molecular mechanisms that underlie their adaption to various environments. However, the isolation of high-quality RNA from these Gram-negative anaerobes is challenging because of their cell wall structure and the limitations of conventional column-based RNA purification kits, particularly their transcript size bias. Here, we present a robust, phenol-based RNA extraction protocol optimized for F. nucleatum that consistently yields high-integrity, DNA-free RNA suitable for transcriptomic analyses, including RNA sequencing (RNA-seq) and reverse transcription quantitative PCR (RT-qPCR). This method enables effective cell lysis, nucleic acid purification, and efficient removal of genomic DNA while preserving all transcript classes, including small regulatory RNAs (sRNAs). This protocol enabled us to successfully isolate RNA from different F. nucleatum strains and the related species Fusobacterium periodonticum and lead to the mapping of transcriptional starts sites and identification of sRNAs. Thus, this protocol provides a basis for future studies investigating the gene expression dynamics and regulatory networks of F. nucleatum under diverse environmental conditions.