16S rRNA sequencing of Mus musculus: fecal samples from pregnant BPH/5 and control C57BL/6 mice
Microbial DNA was extracted from samples using the Qiagen DNeasy PowerSoil extraction kits (Qiagen, USA) according to manufacturers protocol. The V4 variable region of the 16S rRNA gene was amplified with PCR primers 515f/806r in a 30 cycle PCR using the DreamTaq Hot Start PCR Master Mix Kit (Thermoscieniftic, Waltham, MA). PCR was performed in 20 l volumes and included: 2 l (7.5 M concentration) of forward and reverse primers, 12.5 l of Hot Start Taq 2X Master Mix (New England BioLabs Inc., Ipswich, MA., USA), 3.5 l of deionized water, and 2 l of sample DNA. Thermal cycle conditions were 95C for 3 min for the initial denaturing step, followed by 30 cycles of 95C for 30 s, 50C for 1 min, and 72C for 1 min. PCR products were checked on a 2% agarose gel for correct product size formation (approx. 350 bp). Michigan State University Genomics Core performed library preparation prior to Illumina MiSeq sequencing following the manufacturers guidelines (Kozich et al., 2013).