Microbiota of rat: pulp chamber
PCR amplification was performed using fusion primers targeting from V3 to V4 regions of the 16S rRNA gene with the extracted DNA. For bacterial amplification, fusion primers of 341F (5-AATGATACGGCGACCACCGAGATCTACAC-XXXXXXXX-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-CCTACGGGNGGCWGCAG-3; underlining sequence indicates the target region primer) and 805R (5- CAAGCAGAAGACGGCATACGAGAT-XXXXXXXX-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-GACTACHVGGGTATCTAATCC-3). The Fusion primers are constructed in the following order which is P5 (P7) graft binding, i5 (i7) index, Nextera consensus, Sequencing adaptor, and Target region sequence. The amplifications were carried out and the PCR product was confirmed by using 1% agarose gel electrophoresis and visualized under a Gel Doc system. The amplified products were purified with the CleanPCR (CleanNA). Equal concentrations of purified products were pooled together and removed short fragments (non-target products) with CleanPCR. The quality and product size were assessed on a Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA) using a DNA 7500 chip. Mixed amplicons were pooled and the sequencing was carried out at Chunlab, Inc. (Seoul, Korea), with Illumina MiSeq Sequencing system (Illumina, USA) according to the manufacturers instructions.