title: AI2_20um, replicate1
design: Fusobacterium nucleatum is a Gram-negative, spindle-shaped anaerobe that plays a central role in oral biofilm development by bridging early and late colonizers. It is also associated with systemic diseases, including preterm birth and colorectal cancer. It consists of four main subspecies: nucleatum (FNN), polymorphum (FNP), animalis (FNA), and vincentii (FNV). AI-2, a conserved signaling molecule involved in interspecies communication, is synthesized by LuxS, encoded by the luxS gene. While FNA strains produce AI-2, FNN and FNV strains lack luxS. In FNP, luxS is present but truncated by a transposon, rendering it nonfunctional. To test whether FNN can respond to AI-2 despite lacking luxS, we used strain ATCC 23726 and constructed a chromosomal insertion strain (WT::luxS) by integrating the luxS gene from FNA ATCC 51191. Three experiments were performed: (1) comparing WT and WT::luxS strains under anaerobic growth, followed by RNA extraction; (2) treating WT cells at mid-log phase (OD  0.4) with 20 M synthetic AI-2 or vehicle control for 1.5 hours before RNA isolation; and (3) exposing WT cells to culture supernatants from either WT or luxS mutant F. periodonticum ATCC 33693 and collecting RNA after 1.5 hours. These studies aim to determine whether FNN can sense AI-2 and to characterize the transcriptional effects of AI-2 exposure or luxS expression.Total RNA was extracted using the Direct-zol RNA MiniPrep Kit (ZYMO Research, R2051) following the manufacturers protocol. RNA samples were submitted to the Cancer Genomics Center at The University of Texas Health Science Center at Houston (CPRITRP240610) for quality control and sequencing. RNA integrity was assessed using the Agilent RNA 6000 Pico Kit (#5067-1513) on the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), and samples with RNA integrity numbers (RIN) greater than 7 were used for library construction.RNA-seq libraries were prepared from rRNA-depleted RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L, New England Biolabs) in combination with NEBNext Multiplex Oligos for Illumina (E6609S, New England Biolabs), following the manufacturers instructions. Library quality was evaluated using the Agilent High Sensitivity DNA Kit (#5067-4626) on the Bioanalyzer 2100, and concentrations were quantified via qPCR using the Collibri Library Quantification Kit (#A38524500, Thermo Fisher Scientific). ). The libraries were pooled evenly and went for the paired-end 150-cycle sequencing on an Illumina NovaSeq X plus System (Illumina, Inc., USA).