|
| Status |
Public on Aug 20, 2013 |
| Title |
LF/HPP_6_BACUNI |
| Sample type |
RNA |
| |
|
| Source name |
Defined bacterial assemblage of 12 species from a gnotobiotic mouse
|
| Organism |
Bacteroides uniformis ATCC 8492 |
| Characteristics |
mouse: 6
diet group: LF/HPP
|
| Treatment protocol |
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.
|
| Growth protocol |
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.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
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.
|
| Label |
biotin
|
| Label protocol |
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).
|
| |
|
| Hybridization protocol |
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.
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneArray Scanner 7G06.
|
| Description |
17156.BACUNI
Cecal contents collected from a gnotobiotic mouse 42 days after being colonized 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 day 14 and day 28. The 'diet group' field indicates the diet the animal was consuming at the time of sacrifice.
|
| Data processing |
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.
|
| |
|
| Submission date |
Jul 03, 2013 |
| Last update date |
Aug 20, 2013 |
| Contact name |
Nathan P McNulty |
| E-mail(s) |
nathan.p.mcnulty@gmail.com
|
| Phone |
314-362-3963
|
| Organization name |
Washington University School of Medicine
|
| Department |
Center for Genome Sciences and Systems Biology
|
| Lab |
Gordon
|
| Street address |
4444 Forest Park Ave. (5th Floor)
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63108 |
| Country |
USA |
| |
|
| Platform ID |
GPL9803 |
| Series (2) |
| GSE48532 |
Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome (GeneChip) |
| GSE48537 |
Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome |
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