Vibrio vulnificus smcR mutant biofilm treated with QStatin, an inhibitor of the quorum-sensing master regulator SmcR (dupllicates)
Transcriptome analysis confirmed the effects of QStatin, a selective inhibitor of the quorum-sensing master regulator SmcR, on expression of the SmcR regulon. Biofilm cells (WT or smcR mutant V. vulnificus) were grown for 13 hrs at 30°C in polystyrene 6-well plates containing VFMG and QStatin (20 µM) or DMSO (final concentration, 0.04%). After planktonic cells were removed, the biofilms were washed twice with PBS and collected with a cell scraper (SPL, Pocheon-Si, South Korea). The collected biofilm cells were resuspended in PBS and RNAprotect® Bacteria Reagent (Qiagen) was added to stabilize the RNA. Total RNA was isolated using an miRNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. Contaminated DNA was digested with TURBO DNase (Ambion, Austin, TX). The RNA was cleaned up using an RNeasy MinElute Cleanup kit (Qiagen). The quality of the RNA was verified by Chunlab (Seoul, South Korea) using an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano reagents (Agilent Technologies, Waldbronn, Germany). Strand-specific cDNA library construction and RNA sequencing were conducted by Chunlab, Inc. Briefly, mRNA was selectively enriched by depleting ribosomal RNA using a Ribo-Zero rRNA Removal kit (Epicentre, Madison, WI). A cDNA library was constructed from the enriched mRNA using the TruSeq Stranded mRNA Sample Preparation kit (Illumina, San Diego, CA) according to the manufacturer's instructions. The quality of cDNA libraries was evaluated using the same methods used to verify the quality of the total RNA, except that Agilent DNA 1000 Reagents (Agilent Technologies) were used. To ensure biological replication, RNA was isolated from two independent biofilm cells per sample and two libraries were constructed and sequenced. Strand-specific single-ended 50-nucleotide reads from each cDNA library were obtained using HiSeq 2500 (Illumina).