Bacteroides thetaiotaomicron VPI-5482
Protocols: For in vitro RNAseq measurements, 5 mL of bacterial culture were collected at OD600=0.4 from glucose minimal medium, or after 1 hour of carbon starvation. The samples were combined with 10 mL RNAProtect (Qiagen), vortexed, pelleted and stored at -80° C until further processing. For in vivo RNAseq measurements, monocolonized mice were euthanized 6 days after gavage, cecal material was collected, immediately frozen in liquid nitrogen and stored at -80° C until further processing. RNA was purified from cecal samples as described in [Rey, ASBMB 2010] In vitro cultures. Bacteroides thetaiotaomicron VPI-5482 (ATCC 29184) was cultured anaerobically at 37° C in liquid TYG or glucose minimal medium [Martens, Cell Host Microbe 2008] or on brain-heart-infusion (BHI; Becton Dickinson) agar supplemented with 10% horse blood (Quad Five). An anaerobic chamber (Coy Laboratory Products) containing 20% CO2, 10% H2, and 70% N2 was used for all anaerobic microbiology procedures. Individual colonies from BHI-blood agar plates where cultured in amino acid-free minimal medium (0.5% glucose, 100 mM KH2PO4 (pH 7.2), 15 mM NaCl, 8.5 mM (NH4)2SO4, 4mM dithiothreitol (AmericanBio AB00490), 4mM Na2S•9H2O, 5 ng mL 1 haemin chloride, 100 mM MgCl2, 1.4 mM FeSO4•7H2O, 50 mM CaCl2, 1 mg mL 1 vitamin K3, and 5 ng mL 1 vitamin B12) with and without supplementation with 20 amino acids at 0.5 mM for 16 hours, and back-diluted to OD600=0.0005. Bacterial growth was monitored by OD600 measurements with the Biotek Eon Microplate Spectrophotometer. All samples were treated with RNAProtect (Qiagen). Aliquots of ~100 mg of frozen cecal samples were suspended in a solution containing 250 µL of acid-washed glass beads (Sigma-Aldrich), 500 µL of extraction buffer A (200 mM NaCl, 20 mM EDTA), 210 µL of 20% SDS, and 500 µL of phenol:chloroform:isoamyl alcohol (125:24:1, pH 4.5; Ambion), and lysed in a bead beater (BioSpec Products). Cellular debris were removed by centrifugation (8,000 g; 3 min). The extraction was repeated, and the nucleic acids were precipitated with isopropanol and sodium acetate (pH 5.5). The crude extracts were cleaned by RNeasy Mini (Qiagen) with on column DNase I treatment, treated with Baseline-ZERO DNase (Epicentre), and cleaned again by RNeasy Mini. Four RNA extracts per mouse were pooled. The RNA quality was assayed on 2100 Bioanalyzer (Agilent), and only samples with RIN score above 8 were used for RNAseq measurements. rRNA was depleted with Ribo-zero rRNA removal kit for gram-negative bacteria (Illumina). RNAseq library was prepared using ScriptSeq v2 kit (Illumina) with multiplexing, as recommended by the manufacturer.