experiments(N=9 mice in control, N= 10 mice in treatment). All mice were male and of the same size distribution N-acyl serinol metabolite measurement in mouse cecal samples and human stool After withholding IPTG for two weeks, mice from the first experimental set were re-exposed to IPTG in the drinking water for I week to induce hm-NAS gene expression and N-acyl serinol production. Mice were sacrificed and cecal samples taken. Fresh cecal stool from two control mice and two treated mice was resuspended in 5 ml of sterile PBS and extracted 1: I with ethyl acetate. Crude extracts were dried in vacuo and resuspended in methanol normalized by crude extract weight. Each extract was then analyzed by reversed phase liquid chromatography coupled to a 6550 Q-TOF mass spectrometer(Agilent Technologies). Peak identities were confirmed by accurate mass, and also by comparison of chromatographic retention time and MS/MS spectra to those of the purified N-palmitoyl serinol standard. In both mice in the treatment group N-palmitoyl serinol could be detected in the cecal samples whereas N-palmitoyl serinol was detected in neither of the mice in the control group (Extended Data Fig. 6). Stool samples were collected from human subjects prior to bone marrow transplant as part of a previous clinical trial conducted at Memorial Sloan Kettering in collaboration with the author JC. Fresh stool samples were processed in the same manner as the mouse cecal samples described above Data Availability statement All figure data is available in the source data sheet Gene accession num bers for all cloned genes are provided in Supplementary Table l Publically available DNA and rna datasets analyzed in this study are referenced accordingly and references contain links to datasets available for download Nature. Author manuscript; available in PMC 2018 February 28experiments (N = 9 mice in control, N = 10 mice in treatment). All mice were male and of the same size distribution. N-acyl serinol metabolite measurement in mouse cecal samples and human stool After withholding IPTG for two weeks, mice from the first experimental set were re-exposed to IPTG in the drinking water for 1 week to induce hm-NAS gene expression and N-acyl serinol production. Mice were sacrificed and cecal samples taken. Fresh cecal stool from two control mice and two treated mice was resuspended in 5 ml of sterile PBS and extracted 1:1 with ethyl acetate. Crude extracts were dried in vacuo and resuspended in methanol normalized by crude extract weight. Each extract was then analyzed by reversed phase liquid chromatography coupled to a 6550 Q-TOF mass spectrometer (Agilent Technologies). Peak identities were confirmed by accurate mass, and also by comparison of chromatographic retention time and MS/MS spectra to those of the purified N-palmitoyl serinol standard. In both mice in the treatment group N-palmitoyl serinol could be detected in the cecal samples whereas N-palmitoyl serinol was detected in neither of the mice in the control group (Extended Data Fig. 6). Stool samples were collected from human subjects prior to bone marrow transplant as part of a previous clinical trial conducted at Memorial Sloan Kettering in collaboration with the author J.C.. Fresh stool samples were processed in the same manner as the mouse cecal samples described above. Data Availability Statement All figure data is available in the source data sheet. Gene accession numbers for all cloned genes are provided in Supplementary Table 1. Publically available DNA and RNA datasets analyzed in this study are referenced accordingly and references contain links to datasets available for download. Cohen et al. Page 14 Nature. Author manuscript; available in PMC 2018 February 28. Author Manuscript Author Manuscript Author Manuscript Author Manuscript