16S V4 amplicon sequencing of equine uterine
The PCR amplification of extracted DNA and 16S amplicon sequencing was performed by Novogene Corporation Inc, Sacramento, CA. The V4 region of the 16S ribosomal RNA gene (~250bp) was amplified using the specific primer pair 515F (5- GTGCCAGCMGCCGCGGTAA-3) and 806R (5-GGAC TACHVGGGTWTCTAAT-3), with the forward primer carrying the barcode sequence. The Phusion High-Fidelity PCR Master Mix (New England Biolabs) was used to carry out all PCR reactions. The PCR products were purified by running them on a 2% agarose gel and extracting them using a Qiagen Gel Extraction Kit. The sequencing library preparation was done using a NEBNext UltraTM DNA Library Prep Kit for Illumina. It was quality tested using Qubit 3.0 fluorometer and quantified using Q-PCR.