Comment[ArrayExpressAccession] E-GEOD-48537 MAGE-TAB Version 1.1 Public Release Date 2013-08-20 Investigation Title Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome Comment[Submitted Name] Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome Experiment Description This SuperSeries is composed of the SubSeries listed below. Refer to individual Series Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name McNulty Person First Name Nathan Person Mid Initials P Person Email nathan.p.mcnulty@gmail.com Person Affiliation Washington University School of Medicine Person Phone 314-362-3963 Person Address Center for Genome Sciences and Systems Biology, Washington University School of Medicine, 4444 Forest Park Ave. (5th Floor), Saint Louis, MO, USA Person Roles submitter Protocol Name P-GSE48537-1 P-GSE48537-8 P-GSE48537-9 P-GSE48537-2 P-GSE48537-5 P-GSE48537-11 P-GSE48537-13 P-GSE48537-12 P-GSE48537-7 P-GSE48537-4 P-GSE48537-3 P-GSE48537-6 P-GSE48537-10 Protocol Description A custom mask file was first created for each of the 12 species analyzed, after which species-specific Expression Console custom configuration files were prepared (MAS5 probe set summarization; scaling by all select sets specified by probe masking file) and used to execute an advanced analysis. Raw values were then exported as pivot tables, filtered for those probe sets corresponding to the species being analyzed, and passed as input data to the Cyber-T web server. In each treatment group, genes for which at least 5 of 7 samples resulted in a 'present' call for the probe set were scored as having reliably detectable gene expression. ID_REF = VALUE = MAS5.0 signal intensity cDNA was prepared from gDNA-free RNA using random hexamer priming and Invitrogen's SuperScript II enzyme. RNA template was degraded by NaOH treatment, and cDNA was subsequently fragmented by DNase I digestion. Finally, fragmented cDNA was biotinylated using the BioArray Terminal Labeling Kit with biotin-ddUTP (Enzo, 42630). Successful biotinylation of targets was verified using a shift assay with NeutrAvidin (Pierce, 31000). Following fragmentation and labeling, cDNA (0.9-5.1 ug) was hybridized for 16 h at 45 C on custom GeneChip SynComm1 (Synthetic Human Gut Community GeneChip, v1.0, GEO platform GPL9803). GeneChips were washed and stained in the Affymetrix Fluidics Station 450. Immediately after collection, samples were subjected to flash-freezing in liquid nitrogen and storage at -80 C until total community DNA extractions could be performed. Immediately after collection, samples were subjected to flash-freezing in liquid nitrogen and were stored at -80 C until total community RNA could be extracted. Sample was collected from a gnotobiotic mouse colonized at day 0 with a defined assemblage of 12 human gut bacterial species. Each mouse was fed two different diets in an oscillatory fashion, with diet switches occurring at days 14 and 28. This animal was fed a low-fat high-plant polysaccharide (LF/HPP) diet in phase 1 (days 1-14), a high-fat high-sugar (HF/HS) diet in phase 2 (days 15-28) and a low-fat high-plant polysaccharide (LF/HPP) diet in phase 3 (days 29-42). Sample was collected from an in vitro culture in which Bacteroides cellulosilyticus WH2 was grown to early log stage in Bacteroidetes minimal media containing a single carbohydrate as a carbon source. RNAprotect Bacteria Reagent (Qiagen) was used to stabilize both frozen fecal pellets (experiment NM601) and in vitro cultures (experiment NM602; added prior to pelleting cells) per the manufacturer's instructions. Samples were then mechanically disrupted using acid-washed glass beads in a Mini-BeadBeater-8 in the presence of acid phenol:chloroform:IAA (125:24:1), after which ethanol precipitation was performed. RNA was then further purified using a MEGAclear kit, tested for integrity by gel electrophoresis, and shown to be lacking any contaminating genomic DNA by PCR. rRNA depletion was subsequently performed via two rounds of processing with the MICROBExpress kit and two rounds of rRNA capture using custom oligos attached to magnetic beads (Dynabeads). First-strand cDNA synthesis was then performed using random hexamer priming with SuperScript II, after which second-strand synthesis was achieved using E. coli DNA polymerase in the presence of RNase H. Libraries were prepared according to a slightly modified version of the protocol accompanying the Illumina Genomic DNA Sample Prep Kit. Briefly, cDNA was sonicated in a BioRuptor XL water bath sonicator, cleaned up and concentrated using a Qiagen PCR Purification column, and end-repaired using Klenow DNA polymerase. The blunt DNA was treated with Klenow fragment (exo minus) to add an adenine overhang, and the A-tailed molecules were ligated to the relevant Illumina adapter sequence. Non-barcoded adapters were used to construct libraries from experiment NM601 samples. Adapters incorporating an 8 bp barcode were used to construct libraries from experiment NM602 samples. Adaptered-DNA was then size-selected by agarose gel electrophoresis. Fragments of the appropriate size were PCR amplified and purified, after which the purified PCR products were loaded on an Illumina flow cell for cluster generation. Libraries were sequenced on either the Illumina Genome Analyzer IIx (experiment NM601) or the Illumina HiSeq 2000 (experiment NM602) using the manufacturer's protocols. Samples previously stored at -80 C were incubated in Qiagen RNAprotect Bacteria Reagent per the manufacturer's instructions, after which they were mechanically disrupted using acid-washed glass beads in a Mini-BeadBeater-8 in the presence of acid phenol:chloroform:IAA (125:24:1). Following ethanol precipitation, RNA was further purified using the Qiagen RNeasy Mini Kit and subjected to two rounds of DNase treatment. All samples were shown to be free of contaminating gDNA by PCR before RT and labeling were performed. DNA was extracted from fecal and cecal samples using mechanical disruption (bead-beating with 0.1 mm zirconium beads) in phenol:chloroform:IAA in the same manner as described by McNulty et al. in a previous publication (PMID: 22030749). Libraries were prepared according to a slightly modified version of the protocol accompanying the Illumina Genomic DNA Sample Prep Kit. Briefly, total bacterial gDNA was sonicated in a BioRuptor XL water bath sonicator, cleaned up and concentrated using a Qiagen PCR Purification column, and end-repaired using Klenow DNA polymerase. The blunt DNA was treated with Klenow fragment (exo minus) to add an adenine overhang, and the A-tailed molecules were ligated to the relevant barcoded Illumina adapter sequence. Adaptered-DNA was then size-selected by agarose gel electrophoresis. Fragments of the appropriate size were PCR amplified and purified, after which the purified PCR products were loaded on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer I, II or IIx following the manufacturer's protocols. Bacteria from frozen culture stocks were introduced as an artificial community (AC) into 10-12 week-old male gnotobiotic C57BL/6J mice. Cells were therefore maintained over the course of the experiment within mice until sampling occurred. Bacteria from frozen culture stocks were introduced as an artificial community into 10-12 week-old male gnotobiotic C57BL/6J mice. Cells were therefore maintained over the course of the experiment within mice until sampling occurred. GeneChips were scanned using the Affymetrix GeneArray Scanner 7G06. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol sample treatment protocol sample treatment protocol sample treatment protocol nucleic acid library construction protocol nucleic acid library construction protocol nucleic acid library construction protocol growth protocol growth protocol array scanning protocol Experimental Factor Name EXPERIMENT CARBOHYDRATE DIET GROUP DAYS POST-COLONIZATION MOUSE ORGANISM COLLECTION SITE Experimental Factor Type experiment carbohydrate diet group days post-colonization mouse organism collection site Comment[SecondaryAccession] GSE48537 Comment[GEOReleaseDate] 2013-08-20 Comment[ArrayExpressSubmissionDate] 2013-07-03 Comment[GEOLastUpdateDate] 2013-08-21 Comment[AEExperimentType] RNA-seq of coding RNA Comment[AEExperimentType] other Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-48537.hyb.sdrf.txt E-GEOD-48537.seq.sdrf.txt