Faecal samples were collected by the Laboratoire Cœur et Nutrition, TIMC-IMAG CNRS UMR 5525, Université Joseph Fourier, Grenoble, France
Extracted molecule
other
Extraction protocol
Total DNA was extracted from 25 rat faecal samples using Qiagen’s DNA Stool Kit (Qiagen, West Sussex, UK). 10ng of sample was then used for PCR amplification. 16S rRNA genes were amplified using universal primers 27F (AGAGTTTGATCMTGGCTCAG) and 1492R (TACGGYTACCTTGTTACGACT). PCR reactions were performed in a 50µl volume, using DreamTaq DNA polymerase (Fermentas, St. Leon-Rot, Germany). The PCR reaction consisted of an initial denaturation step at 95°C for 5min followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C for 40 s and elongation at 72°C for 2 min. A final extension step was performed at 72°C for 5 min. PCR product size was verified by electrophoresis with 1% (w/v) agarose gel and were purified using the MinElute PCR Purification Kit (Qiagen Ltd., UK) following manufacturer’s instructions and stored at -20°C.
Label
Cy3
Label protocol
For each sample, the purified 16S rRNA gene PCR products (1 µg) were labelled with either Cy3 or Cy5 using the Genomic DNA ULS labelling Kit (Agilent Technologies, Palo Alto, CA) following the manufacturer’s instructions.
Hybridization protocol
For microarray hybridization, 250ng of each labelled faecal sample were used. Hybridization was performed following the Agilent OligoaCGH hybridization protocol (Agilent Technologies, Palo Alto, CA) at 65°C for 24h.
Scan protocol
slides were scanned at a 3-µm resolution using a Surescan microarray scanner (Agilent Technologies, Palo Alto, CA).
Description
LO3 raw data file: LO3_Cy3_And_LO+A1_Cy5.txt 16S rRNA gene amplified from gDNA extracted from faecal samples of treated rats.
Data processing
Pixel intensities were extracted using the 'Feature Extraction' software (Agilent Technologies, Palo Alto, CA). No normalization step was performed and the retained intensity value for each probe was the ratio between the spot’s median intensity signals and the median of background signals.
