Different GH cloned in plasmid or fosmid (pDest vector) were expressed by recombinant E. coli strains as previously described , (Cecchini et al., 2013; Bastien et al., 2013; Arnal et al., 2015, Vincentelli et al., 2011). (Tasse et al., 2010; Song et al., 2012;Ladevèze et al., 2013;). Briefly, cultures were stopped at OD600nm between 0.4 and 0.6, and cells were harvested by centrifugation for 10 minutes at 5000 rpm and 4°C. The supernatant was then discarded and the bacterial pellet immediately frozen at -80°C before RNA extraction. Microbial consortia analyzes were performed on an anaerobic rumen-derived consortium RWS, which efficiently degrades lignocellulose, as reported by Lazuka et al. (Lazuka et al., 2015).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from bacterial pellets after lysis with 1mg/ml lysozyme (Sigma-Aldrich, Isle d'Abeau Chesnes, France) during 5 min at 25°C, using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France) following the manufacturer's instructions. RNA concentration, purity and integrity was evaluated by measuring the absorbance ratio at 260/280 nm and 260/230 nm using a Nanodrop spectrophotometer (Labtech, Palaiseau, France). The Ratio Integrity Number (RIN) was evaluated using 2100 Bioanalyzer® (Agilent Technologies, Massy, France) and only samples with a RIN upper than 8 were hybridized on the microarray. Total RNA of rumen derived consortium was extracted in two steps from nitrogen frozen samples using the PowerMicrobiome RNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA) (Lazuka et al., 2015). RNA purification was performed using AllPrep DNA/RNA minikit (Qiagen), following the manufacturer’s instructions.
Label
Cy3
Label protocol
The One-Color Low Input Quick Amp WT Labeling Kit™ (Agilent Technologies, Massy, France) was used to amplify and label 100ng of RNA following the manufacturer's instructions. The efficiency of the labeling was checked using a NanoDrop spectrophotometer operating at 260 nm to quantify cRNA and at 550 or 660 nm to measure cyanine 3 (Cy3) and cyanine 5 (Cy5) dye incorporation respectively. Labeling efficiency was calculated as indicated by the manufacturer’s protocol (ratio cyanine quantity / amount of RNA) and was above 6.
Hybridization protocol
For each sample, 1650 ng of labeled and amplified cRNA was used for hybridization. The hybridization master mix was prepared according to manufacturer’s protocol (Agilent Technologies, Massy, France) and 100 µl were dispensed onto a gasket slide, according to the Agilent Microarray Hybridization Chamber User Guide. Afterwards, the active side of the microarray slide was placed on top of the gasket to form a properly aligned “sandwich slide pair”. The microarray slides were inserted into the hybridization chamber (Agilent Technology) than placed at 65°C for 17 hours with rotation at 10 rpm. After hybridization, the microarray was washed over a 1-min period first using Gene Expression Wash Buffer 1 and then Gene Expression Wash Buffer 2 (Agilent Technologies, Massy, France) pre-warmed at 37°C.
Scan protocol
After washing, the arrays were immediately scanned using an MS200 scanner (NimbleGen Roche Diagnostics, Meylan, France) with NimbleGen MS200 software v1.2 at 2 micron resolution.
Description
gene expression of plasmidic GH
Data processing
The median signal of each spot in the hybridized arrays was determined and quantified using Feature Extraction software v11.5.1.1. The data from all the microarrays were normalized together using the “limma” package function “normalizeQuantiles” and the “quantile” method (Bolstad et al., 2003; Smyth et al., 2005). Normalization and statistical analyses of the data were performed using the Bioconductor packages (http://www.bioconductor.org) and R software v3.1.3. The normalized fluorescence intensities of three experimental replicates per sample were analyzed and the mean values, standard deviations and correlation coefficients (%CV) were calculated. To determine whether probes were specific and target genes present, limma one way ANOVA tests were carried out using the False Discovery Ratio method and an adjusted p value < 0.05. Limma t test using limma package, was conducted to know in which gene is differentially expressed.
