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| Status |
Public on Aug 30, 2016 |
| Title |
Gut_microbiome_AB1 |
| Sample type |
genomic |
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| Source name |
human fecal sample, healthy with alcohol abuse
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| Organism |
human metagenome |
| Characteristics |
disease state: healthy with alcohol abuse
gender: Male
age: 31
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fecal samples were immediately frozen on collection and stored at -80℃ before analysis. A frozen aliquot (200 mg) of each fecal sample was added to a 2.0-ml screwcap vial containing 300 mg glass beads of 0.1 mm diameter (Sigma, St. Louis, MO, USA), and kept on ice until the addition of 1.4-ml ASL buffer from the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA, USA). Samples were immediately subjected to beadbeating (45 s, speed 6.5) using a FastPrep machine (Bio 101, Morgan Irvine, CA, USA), prior to the initial incubation for heat and chemical lysis at 95uC for 5 minutes. Subsequent steps of DNA extraction followed the QIAamp kit protocol for pathogen detection.
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| Label |
Cy3
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| Label protocol |
For each sample, 1 μg of DNA was labeled with the fluorescent dye Cy-3 (GE Healthcare, CA, USA) using random primers and the Klenow fragment of DNA polymerase I (Wu et al. 2006). Labeled DNA was then purified using a QIAquick Purification kit (Qiagen, Valencia, CA, USA), dried in a SpeedVac at 45℃ for 45 min (Thermo Savant, NY, USA). Dried DNA was then rehydrated with 13 μL of DNase/RNase-free distilled water, mixed completely, and centrifuged to collect all liquid at the bottom of the tube. A total of 42 μL of buffer, including 1× HI-RPM hybridization buffer,1× aCGH blocking agent, 0.05 μg•μL−1 Cot-1 DNA, 10 pM universal standard, and 10% formamide (final concentrations), was added to each sample. After mixing completely by vortexing, the solution was spun down and incubated at 95°C for 3 min, then incubated at 37°C for 30 min.
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| Hybridization protocol |
The samples were then hybridized with HuMiChip2 at 67°C for 24 h with a rotation at 20 rpm in an Agilent hybridization oven (Agilent Technologies, Inc., CA Santa Clara, CA, USA).
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| Scan protocol |
Arrays were scanned at full laser power and 100% photomultiplier tubes gain with a NimbleGen MS 200 Microarray Scanner (Roche NimbleGen).
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| Data processing |
The scanned images of hybridized HuMiChip2 were converted and extracted using the Agilent Feature Extraction 11.5 software (Agilent Technologies, Inc., CA, USA) for further data analysis. Probe spots with coefficient of variance (CV) greater than 0.8 were removed. Probes with SNR (signal-to-noise ratio) less than 2 and signal intensities less than 500 were also removed. Microarray data was then normalized based on the total signal intensity of CORS (common oligonucleotide reference standard) probes (Liang et al. 2010).
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| Submission date |
Aug 29, 2016 |
| Last update date |
Aug 30, 2016 |
| Contact name |
Qichao Tu |
| E-mail(s) |
philloid@gmail.com
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| Organization name |
University of Oklahoma
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| Street address |
101 David L Boren Blvd
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| City |
Norman |
| State/province |
OK |
| ZIP/Postal code |
73019 |
| Country |
USA |
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| Platform ID |
GPL22376 |
| Series (1) |
| GSE86162 |
HuMiChip2 for Strain Level Identification and Functional Profiling of Human Microbiomes |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM2296243_256199110035_a01_532_635_converted_CGH_1105_Oct12_1_4.txt.gz |
49.8 Mb |
(ftp)(http) |
TXT |
| Processed data are available on Series record |
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