title: Sample BMS612, Obese Animal 2259, Luteal Phase, Maintenance, Cycle 9 Day 4
design: DNA was extracted from the vaginal swabs and cervical flushings of all pigs using the PowerSoil ¨ DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturerÕs instructions. After extraction the DNA stock concentration was measured using a NanoDrop-2000 spectrophotometer (Thermo Scientific, Inc., Wilmington, DE, USA), with samples ranging from 12.6 to 510 ng/_l. A 584 bp fragment of the hypervariable V3-V5 region of the 16S rRNA gene was amplified by a polymerase chain reaction (PCR). The 357F forward primer was used (read 1: 5ÕTATGGTAATTGTCCTACGGGAGGCAGCAG3Õ; read 2: 5ÕAGTCAGTCAGCCCCGTCAATTCMTTTRAGT3Õ; index: 5ÕACTYAAAKGAATTGACGGGGCTGACTGACT3Õ). The universal reverse primer was 926R (5ÕCCGTCAATTCMTTTRAGT3Õ). One of 96 specific barcodes consisting of 12 base pairs was also added to the reverse primer. The PCR products were then run on an agarose gel, where it was determined that a nested PCR amplification was required. Following the nested PCR, the products were then purified using the GeneJET PCR Purification Kit (Thermo Scientific, Inc., Wilmington, DE, USA). Final DNA product concentrations were measured by the NanoDrop-2000 spectrophotometer; samples were then pooled together by mass (15 _g), with one pool consisting of samples containing reverse primer barcodes 1-96. Total pool concentration was measured by Qubit 2.0 Fluorometer (Invitrogen, Life Technologies, Carlsbad, CA, USA). All pools were then sent to the University of Illinois at Urbana-Champaign to be sequenced utilizing a high-throughput platform (MiSeq; Illumina Inc., San Diego, CA,USA). Approximately 87,000,000 total sequence reads with 50,000 reads/sample were obtained, including both dominant and poorly-represented taxa of the urogenital microbiome.