16S rRNA sequencing of ex-vivo oral microbiomes: propagation in CDM or SHI medium
16S sequences were PCR amplified with primers sIDTP_515F and sIDTP7_806R (CTACACGACGCTCTTCCGATCTGTGCCAGCMGCCGCGGTAA and CAGACGTGTGCTCTTCCGATCTGGACTACHVGGGTWTCTAAT  underlined regions represent linker sequences) respectively. ). Amplicons were generated using a two-stage PCR amplification protocol. The primers contained 5 common sequence tags, underlined (sIDTP5 and sIDTP7) that match 3 sequences present in IDT xGen Amplicon UDI primers (part numbers: 10009846, 10009851, 10009852, and 10009853). First stage PCR amplifications were performed in 10 microliter reactions in 96-well plates, using repliQa HiFi ToughMix (Quantabio). Genomic DNA input was 4 microliters per reaction. PCR conditions were 98C for 2 minutes, followed by 28 cycles of 98C for 10, 52C for 1 and 68C for 1. Subsequently, a second PCR amplification was performed in 10 microliter reactions in 96-well plates using repliQa HiFi ToughMix. Each well received a separate primer pair containing unique dual indices (i.e., from the IDT xGen amplicon UDI primer sets). One microliter of PCR product from the first stage amplification was used as template for the 2nd stage, without cleanup. Two microliters of primer were used per reaction. Cycling conditions were 98C for 2 minutes, followed by 10 cycles of 98C for 10, 60C for 1 and 68C for 1. The PCR product was pooled prior to a 0.6X Ampure cleanup. The pooled products were cleaned as described above (i.e., Pippin Prep and Ampure cleanups) and sequenced on an Illumina NovaSeq6000 instrument using a 500-cycle SP flow cell with read lengths extended to 2x259 bases. Library preparation, pooling, and MiniSeq sequencing were performed at the Genomics and Microbiome Core Facility (GMCF) at Rush University. NovaSeq sequencing was performed at the DNA Services Facility at the Roy J. Carver Biotechnology Center at the University of Illinois at Urbana-Champaign.