normalization data transformation protocol
Microarray data was processed using limma package in R 2.15.1. Loess within array normalisation and Aquantile between array normalisation was applied, without background correction. ID_REF = VALUE = normalized log2 ratios of Ch1 (Cy3) / Ch2 (Cy5)
array scanning protocol
The microarray slides were scanned using a DNA Microarray Scanner G2565CA (Agilent Technologies) according to the manufacturer’s instructions.
The labelled samples were hybridized onto Agilent Technologies 44k whole human genome oligonucleotide arrays (G4112A) according to the manufacturer’s instructions (Two-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling), Version 5.7, March 2008, Agilent Technologies).
The RNA samples were amplified and labeled with either cyanine-3 or cyanine-5 labeled CTP dye using the Agilent Quick-Amp Labeling Kit (5190-0424) as per the manufacturer's instructions (Two-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling), Version 5.7, March 2008, Agilent Technologies).
sample treatment protocol
After 14 days the inserts were transfered to a custom built co-culture chamber placed inside an anaerobic workstation, thus exposing the Caco-2 cells to an anaerobic environement (10% CO₂, 10% H₂ in N₂) on the apical side, while maintaining an aerobic basal side (apical anaerobic conditions). The Caco-2 cells were co-cultured with Faecalibacterium prausnitzii DSM17677, Lactobacilus rhamnosus HN001, UV-killed F. prausnitzii, or no bacteria in the apical side for four hours. Bacteria were added at a multiplicity of infection of 100.
nucleic acid extraction protocol
RNAprotect Cell Reagent (Qiagen) was added at a volume of approximately nine times the volume of the apical media to lyse the Caco-2 cells and stabilize the RNA. Total RNA was extracted from the Caco-2 cells using an RNeasy mini plus kit (Qiagen) as per the manufacturer’s protocol.
Caco-2 cells were cultured in Transwell inserts in Medium 199 (M199) containing 10% (v/v) fetal bovine serum, 1% (v/v) nonessential amino acids (NEAA; 100x solution) and 1% Penicillin-Streptomycin (10,000 U penicillin G sodium salt and 10,000 mg streptomycin sulphate in 0.85% (v/v) saline) at 37°C in 5% CO₂.