16S vaginal microbiome of Equus caballus
DNA from samples was extracted using the DNeasy PowerLyzer PowerSoil Kit (Qiagen, Hilden, Germany) following manufacturers instructions. Samples were amplified using primers specific to the V3-V4 regions of the 16S rRNA DNA. PCR products were purified using AMPure XP beads (Beckman Coulter, Nyon, Switzerland) with a 0.9 ratio according to the manufacturers instructions. PCR products were eluted from the magnetic beads with 32 L of Milli-Q water and 30 L of the eluate were transferred to a fresh 96-well plate. The above-described primers contain overhangs allowing the addition of full-length Nextera barcoded adapters for multiplex sequencing in a second PCR step, resulting in sequencing ready libraries with approximately 450 bp insert sizes. In brief, 5 L of the first PCR purified product were used as the template for a second PCR with Nextera XT v2 adaptor primers in a final volume of 30 L using the same PCR mix and thermal profile as for the first PCR but with only 8 cycles. 25 L of the second PCR product were purified with SequalPrep normalization kit (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturers protocol. Libraries were eluted in 20 L final volume and pooled for sequencing.