Omni bead ruptor 24

Mouse lungs were homogenized using ceramic beads (Omni Inc, Kennesaw, Georgia) and the Omni Bead Ruptor 24 (Omni Inc, Kennesaw Georgia). Total RNA was isolated from homogenized mouse lung using Trizol (Invitrogen, Carlsbad, CA) and RNA was purified using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. DNase treatment was performed (Qiagen, Valencia, CA) before being reverse transcribed with SuperScript™ IV Vilo Master Mix™ (Invitrogen, Carlsbad, CA). Reverse-Transcriptase quantitative PCR (RT-qPCR) analysis was done using LightCycler FastStart DNA master SYBR green I as a ready-to-use reaction mixture (Roche, Indianapolis, Indiana). Targeted genes were amplified from cDNA using the primers listed in Supplementary Table S11 and gene expression was normalized to Rpl13a (mouse) or GAPDH (human). Melting curves have been generated for all assays and one product for each gene has been identified.

Genomic DNA of PolyFermS microbiota was extracted with the FastDNA® SPIN Kit for soil (MP Biomedicals Europe, Illkirch, France) following the protocol of the supplier. Pellets of 2 ml PolyFermS effluent were resuspended in MT lysis buffer and disrupted in Lysing Matrix E tubes with the Omni Bead Ruptor 24 (OMNI International, Kennesaw, United States) at 16 m s⁻¹ for 40 s. Quality of genomic DNA was assessed by 1.5% (m/v) agarose electrophoresis and concentration was determined by Nanodrop ND-100 Spectrophotometer (Thermo Fisher Scientific, Wilmington, USA). DNA samples were diluted in nuclease-free water to 20 ng μl⁻¹ and stored at 4 °C until further processing. The V4 region of the 16S rRNA gene was amplified by PCR with forward 515F (GTG CCA GCM GCC GCG GTA A) and reverse 806R (GGACT ACH VGG GTW TCT AAT) primer (Caporaso et al. 2011). Negative controls with PCR water as template were included. Library preparation and sequencing was conducted at the Genetic Diversity Center (GDC, ETH Zurich, Switzerland) following a previously described protocol for library preparation [78 (link)]. Sequencing was performed using an Illumina MiSeq flow cell with V2 2 × 250 bp paired end chemistry supplemented with 20% of PhiX.

Two-milliliter Tough Tubes with caps, ceramic beads 1.4 mm and 2.8 mm, and Omni Bead Ruptor 24 were purchased from Omni International, Kennesaw, GA, USA. Pipette tips (200 µL Genomic LR (orifice diameter 1.5) low retention) were purchased from VWR International, Radnor, PA, USA. Data were integrated with Lab Solutions 5.97 Shimadzu Corporation, Kyoto, Japan, Lab Solutions Insight LCMS. Pierce™ BCA Protein Kit was obtained by Thermo Scientific, Waltham, MA, USA. LC-MS grade isopropanol, methanol, and acetonitrile were obtained from Fischer Chemicals, Pittsburgh, PA, USA. Taurocholic acid, glycocholic acid, chenodeoxycholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, and their corresponding deuterium-labeled compounds, as well as hydrochloric acid 37%, ammonium formate, and formic acid were purchased from Sigma-Aldrich, Burlington, MA, USA.