Microbiome Profiling of Dental Plaque

For each individual, a supragingival plaque sample was collected from all surfaces of all teeth by using a sterilized toothpick. Professional tooth cleaning was stopped for 1 month, and patients were instructed to stop their own plaque control at least 1 h before the sample collection. The collected plaque was placed into PM1 buffer in the PowerMicrobiome RNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) in a sterile tube, and was stored at − 80 °C. The RNA extraction, cDNA synthesis, library preparation, and Illumina sequencing were conducted, as described previously [18 (link)] with the following modifications. The SuperScript® Double-Stranded cDNA Synthesis kit (Thermo Fisher Scientific, Waltham, MA, USA) and 6-mer random primer were used instead of the SMARTer Ultra Low RNA kit (Clontech, Mountain View, CA, USA). MiSeq (Illumina, Inc., San Diego, CA, USA) reads were generated as 300-bp paired-end.