K2 edta plasma tubes

Blood was collected at baseline via cardiac puncture into K2 EDTA Plasma tubes (Sarstedt). Red blood cells were lysed with ACK lysis buffer, cells were washed, gently pelleted and resuspended in 500 μl of TRIzol (Invitrogen Life Technologies). RNA was extracted using TRIzol according to manufacturer’s instructions. rRNA was removed using the NEBNext rRNA Depletion Kit (NEB), followed by cDNA library preparation using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). The libraries were sequenced on illumina HiSeq 3000 and the sequenced reads were aligned to the mouse genome using TopHat (65 (link), 66 (link)). Cufflinks and Cuffdiff were used to quantify transcripts and determine differential expression (65 (link), 66 (link)). Further analysis and visualization were performed using PARTEK and R.

Gut microbiota analysis was based on cecal material, unless otherwise stated. Mucosal-associated and luminal microbiota were harvested from cecal material as previously described (15 (link)). Fecal pellets were collected for some experiments as indicated (Fig. 5 and Fig. S5). To harvest skin microbiota, ears were excised from three mice under sterile conditions and pooled into a sterile DNA-free Eppendorf Biopur® tube containing 600 μl of PBS. This was followed by shaking at 1,000 rpm and 37°C for 30 min and centrifugation at 5,000 × g for 10 min. After removal of the supernatant, pellets were snap-froze and stored at −80°C. To harvest vaginal microbiota, vaginas were carefully swabbed through a gentle rotating motion to reduce host DNA contamination, while avoiding any contamination with skin microbiota (COPAN Diagnostics Inc., FLOQSwab certified DNA free). Vaginal samples were processed identically to skin samples with the exception of shaking at 20°C. All samples were frozen at −80°C until use. Blood was drawn via cardiac puncture and processed in either Serum micro tubes Z-Gel (Sarstedt) or K2 EDTA Plasma tubes (Sarstedt) according to manufacturer recommendations, snap-frozen, and stored at −80°C.