16S seq of mouse larynx
V4 regions of the 16S rRNA gene were amplified using MolTaq 16S/18S DNA-free Taq Polymerase (Molzym GmbH & Co. KG, Bremen, Germany) in a 25 ml reaction containing 10 ng of DNA template and 200 nM of 515F/806R primers fused with Illumina sequencing adapters.77 Non-template control (NTC, n=3) and a PCR positive control with Helicobacter pylori genomic DNA as template was included for each PCR run. PCR cycling conditions were as follows: one cycle of enzyme activation at 95_x000E_C for 3 min followed by 35 cycles of denaturation at 95_x000E_C for 30 sec, annealing at 55_x000E_C for 30 sec, and extension at 72_x000E_C for 30 sec, and a final extension at 72_x000E_C for 5 min. Each sample was amplified in triplicate. Resulting PCR products were identified on 2% agarose gel, and the successful reactions were pooled, purified with NucleoSpin Gel and PCR cleanup kit (Takara, San Jose, CA), and quantified with Qubit dsDNA HS Assay Kit (Invitrogen, Eugene, OR). Samples were equimolar pooled and sequenced on the Illumina MiSeq 2x250 bp platform.