title: Illumina HiSeq 4000 paired end sequencing; Effects of microbial disruption on intestinal macrophages.
design: Effects of microbial disruption on intestinal macrophages.
library_construction_protocol: Mice received an antibiotic cocktail containing ampicillin (1g/liter), metronidazole (1g/liter), neomycin (1g/liter), gentamicin (1g/liter), and vancomycin (0.5g/liter) in drinking water for 7 days, which was replaced once, after 3 to 4 days. Mice were administered acetate, propionate, or butyrate in their drinking water for 7 days at a concentration of 200 mM. SCFAs were added to LP cells in vitro at a concentration of 1 mM for 4 hours. These concentrations of SCFAs were based on published work (15, 17) and pilot experiments (no in vivo effects on macrophages <100 mM and decreased oral intake >300 mM; in vitro concentrations assessed for efficacy versus toxicity). The administered in vivo amount of 200 mM is higher than the physiological concentrations in the colon (20 to 50 mM) to take into account the absorption in the small intestine that will reduce the amount of SCFA reaching the colon. For in vitro experiments, 1 mM butyrate was used to take into account amounts of butyrate that might reach macrophages in the LP from the colonic lumen, where it is known that a substantial amount would be taken up by epithelial cells. Feces from control mice (housed separately from experimental mice) were administered to cages of both the experimental control group and the experimental antibiotic-treated group. Specifically, sawdust and feces from several cages of untreated (non-experimental) control mice were mixed together and distributed evenly between the experimental control group and the experimental antibiotic-treated group. Thus, mice were recolonized for variable time periods after 7-day antibiotic treatment. Cages and feces were replaced every 7 days. Intestinal macrophages were collected using an Aria Fusion FACS (fluorescence activated cell sorting) machine, from digested intestinal tissues. Macrophages were identified as CD45+ live CD11b+CD64+MHC Class II+ Ly6C- cells following neutrophil (Ly6G) and eosinophil (SiglecF) exclusion.  RNA was extracted from samples using the Qiagen micro-Plus kit, following isolation of macrophages (via FACS) which were collected in RLT buffer. TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer's protocol
attributes.tag: Experimental Factor: compound
attributes.value: microbiota
attributes.tag: Experimental Factor: dose
attributes.value: 1