title: 16S vaginal microbiome of Equus caballus
design: 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.