Yano et al Page 12 GI Transit Assay Mice were orally gavaged with 200 ul sterile solution of 6% carmine red (Sigma Aldrich) and 0.5% methylcellulose(Sigma Aldrich)in water, and placed in a new cage with no bedding Li et al., 2011). Starting at 120 minutes post-gavage, mice were monitored every 10 minutes for production of a red fecal pellet. GI transit time was recorded as the total number of minutes elapsed (rounded to the nearest 10 minutes) before production of a red fecal pellet For mice treated intrarectally with PCPA or metabolites, GI transit assay was ne third inj Platelet Activation and Aggregation Assays Blood samples were collected by cardiac puncture, diluted with a 2x volume of HEPES medium and centrifuged through PST lithium hepararin vacutainers(Becton Dickinson) Expression of platelet activation markers was measured by flow cytometry(Nieswandt et 于93 al., 2004, Ziu et al., 2012). Platelet aggregation assays were conducted according to methods described in(De Cuyper et al., 2013). Remaining unstained PRP was used to generate PRP smears. Slides were stained with Wright Stain( Camco)according to standard procedures 16S rRNA Gene Sequencing and Analysis Fecal samples were collected at two weeks after orally gavaging GF mice with Sp or hSp Bacterial genomic DNA was extracted from mouse fecal pellets using the QlAamp DNA Stool Mini Kit( Qiagen). The library was generated according to methods from( Caporaso et al., 2011). The V4 regions of the 16s rRNA gene were PCR amplified, purified and then chosen de novo with UPARSE pipeline( Edgar, 2013). Taxonomy assignment andwere sequenced using the Illumina Miseq platform Operational taxonomic units(OTUs) rarefaction were performed using QIIMEl.8.0( Caporaso et al., 2010). Phylogenetic trees were built using Phy ML (Guindon et al., 2010)and visualized using iToL ( Letunic and Bork,2007) Supplementary Materia Refer to Web version on PubMed Central for supplementary material ACKNOWLEDGMENTS The authors acknowledge the assistance of Taren Thron, Sara Mc Bride and Alyssa Maskell for caring for the Dr. Nathan Dalleska and Jesse Allen( Caltech) for conducting pilot LC/MS experiments, Said bogatyrev( Caltech) for helpful advice, Natasha Shelby( Caltech) for editing the manuscript and the late Dr. Paul H. Patterson for his valuable support. This work was supported by the NIH Director's Early Independence Award(5DP5OD017924, to E.Y. H ) Caltech Center for Environmental Microbial Interactions Award (to E.Y. H), NSF Emerging Frontiers i Research and Innovation Award (EFRl-1 137089; to R.F. I and s.K. M ), NHGRI grant(ROIHGO05826; toR..I), NIDDK grant(DK078938: to S.K. M )and NIMH grant (MH100556; to S.K. M) REFERENCES Alemi F, Poole DP, Chiu J, Schoonjans K, Cattaruzza F, Grider JR, Bunnett NW, Corvera CU. The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice. Gastroenterology. 2013: 144: 145-154[ PubMed: 23041323 Cell. Author manuscript; available in PMC 2016 April 09GI Transit Assay Mice were orally gavaged with 200 ul sterile solution of 6% carmine red (Sigma Aldrich) and 0.5% methylcellulose (Sigma Aldrich) in water, and placed in a new cage with no bedding (Li et al., 2011). Starting at 120 minutes post-gavage, mice were monitored every 10 minutes for production of a red fecal pellet. GI transit time was recorded as the total number of minutes elapsed (rounded to the nearest 10 minutes) before production of a red fecal pellet. For mice treated intrarectally with PCPA or metabolites, GI transit assay was conducted 1 hour after the third injection. Platelet Activation and Aggregation Assays Blood samples were collected by cardiac puncture, diluted with a 2× volume of HEPES medium and centrifuged through PST lithium hepararin vacutainers (Becton Dickinson). Expression of platelet activation markers was measured by flow cytometry (Nieswandt et al., 2004; Ziu et al., 2012). Platelet aggregation assays were conducted according to methods described in (De Cuyper et al., 2013). Remaining unstained PRP was used to generate PRP smears. Slides were stained with Wright Stain (Camco) according to standard procedures. 16S rRNA Gene Sequencing and Analysis Fecal samples were collected at two weeks after orally gavaging GF mice with Sp or hSp. Bacterial genomic DNA was extracted from mouse fecal pellets using the QIAamp DNA Stool Mini Kit (Qiagen). The library was generated according to methods from (Caporaso et al., 2011). The V4 regions of the 16S rRNA gene were PCR amplified, purified and then sequenced using the Illumina MiSeq platform. Operational taxonomic units (OTUs) were chosen de novo with UPARSE pipeline (Edgar, 2013). Taxonomy assignment and rarefaction were performed using QIIME1.8.0 (Caporaso et al., 2010). Phylogenetic trees were built using PhyML (Guindon et al., 2010) and visualized using iTOL (Letunic and Bork, 2007). Supplementary Material Refer to Web version on PubMed Central for supplementary material. ACKNOWLEDGMENTS The authors acknowledge the assistance of Taren Thron, Sara McBride and Alyssa Maskell for caring for the animals, Andrew Stefka and Taylor Feehley (University of Chicago) for contributing pilot serum and fecal samples, Dr. Nathan Dalleska and Jesse Allen (Caltech) for conducting pilot LC/MS experiments, Said Bogatyrev (Caltech) for helpful advice, Natasha Shelby (Caltech) for editing the manuscript and the late Dr. Paul H. Patterson for his valuable support. This work was supported by the NIH Director’s Early Independence Award (5DP5OD017924; to E.Y.H.), Caltech Center for Environmental Microbial Interactions Award (to E.Y.H.), NSF Emerging Frontiers in Research and Innovation Award (EFRI-1137089; to R.F.I. and S.K.M.), NHGRI grant (R01HG005826; to R.F.I.), NIDDK grant (DK078938; to S.K.M.) and NIMH grant (MH100556; to S.K.M.). REFERENCES Alemi F, Poole DP, Chiu J, Schoonjans K, Cattaruzza F, Grider JR, Bunnett NW, Corvera CU. The receptor TGR5 mediates the prokinetic actions of intestinal bile acids and is required for normal defecation in mice. Gastroenterology. 2013; 144:145–154. [PubMed: 23041323] Yano et al. Page 12 Cell. Author manuscript; available in PMC 2016 April 09. Author Manuscript Author Manuscript Author Manuscript Author Manuscript