title: Illumina HiSeq 2000 sequencing; RNAseq of the human gut commensal Bacteroides thetaiotaomicron wildtype, relA (bt0700) and ppGpp0 (bt0700 bt3998) strains across three conditions: glucose minimal medium, 1h of carbon starvation, and mouse gut colonization
design: RNAseq of the human gut commensal Bacteroides thetaiotaomicron wildtype, relA (bt0700) and ppGpp0 (bt0700 bt3998) strains across three conditions: glucose minimal medium, 1h of carbon starvation, and mouse gut colonization
library_name: WT_mouseCecum_2_s
library_construction_protocol: 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] Mouse colonization. All experiments using mice were performed using protocols approved by the Yale University Institutional Animal Care and Use Committee. Germ-free Swiss Webster mice were maintained in plastic gnotobiotic isolators with a 12 hours light/dark cycle. Individually caged animals (n = 5/group) were fed a standard, autoclaved mouse chow (5K67 LabDiet, Purina). Barcoded bacterial strains were cultured individually for 16 hours in glucose minimal medium. For monocolonization assays, animals were colonized with 10^8 CFUs of single bacterial culture, fecal samples were collected over the time course of 6 days and frozen immediately at -80° C. Total gDNA was recovered from the gavage material and fecal samples, and strain abundances were determined using the barcode qPCR assay [Cullen, Science 2015].  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.
attributes.tag: Experimental Factor: genotype
attributes.value: wild type genotype
attributes.tag: Experimental Factor: growth condition
attributes.value: mouse cecum