lifestyle: Sessile associated to Artificial roots daypostinoculation: 7
Growth protocol
Before inoculation, Azospirillum lipoferum 4B cells were grown overnight in Nfbm medium (Vial et al. J. Bacteriol. 108:5364-75). Bacterial cells were harvested in the late-exponential phase (at OD580 around 1.2), centrifuged and resuspended in a defined volume of 0.8% NaCl to obtain 2 x109 cells mL-1. Then, 109 cells were mixed with 50 ml plant agar (8 g l-1) and introduced into 120 × 120 × 17 mm square plates.All the plates were incubated vertically, for 7 d in a growth chamber (MLR350, SANYO, UK) with a photoperiod of 16 h at 28°C (light 150 μE m-2 s-1), and 8 h at 22°C in the dark.
Extracted molecule
total RNA
Extraction protocol
For each condition, the bacterial cell pellet was resuspended in 960 µl of suspension buffer (Environ. Microbiol. 14:2645-2660) and transferred in 1.5 ml tubes containing 400 mg of glass beads (Sigma). Cell lysis was realized by shaking twice for 1 min with the TissueLyser II equipment (Qiagen); after a centrifugation of 5 min, at 4°C, 13,000 rpm, the aqueous phase containing ribonucleic acids was taken, and 1 ml of TRIzol® Reagent (Invitrogen, Carlsbad, CA, USA) was added. After incubation of 5 min at room temperature, 100 µl of phenol/chloroform/isoamyl alcohol (25:24:1) were added, the samples were homogenized, incubated during 5 min at room temperature and centrifuged for 10 min, at 4°C, 13,000 rpm. A second phenol/chloroform/isoamyl alcohol extraction (200 µl) was done and ribonucleic acids were precipitated overnight at -20°C in a solution containing 2 volumes of 100 % ethanol, 0.1 volume of 7.5 M ammonium acetate and 0.01 volume of 5 mg ml-1 glycogen. Samples were centrifuged during 15 min, at 4°C, 13,000 rpm and the pellets were rinsed twice with 70 % ethanol before resuspension. PCR (16S rrna) on all samples confirmed that the DNase I (Invitrogen) treatment had removed all remaining DNA. RNA integrity was assessed using Agilent RNA 6000 Pico Kit (Agilent Technologies, Waldbronn, Germany) and the Agilent 2100 Bioanalyzer (Agilent Technologies) device.
Label
Cy3
Label protocol
Labeling was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Hybridization protocol
Hybridization was performed by NimbleGen Systems Inc., Madison, WI, USA following their standard operating protocol. See www.nimblegen.com.
Scan protocol
Scanner model is GenePix 4000B.
Description
This sample is the first replicate of Azospirillum lipoferum 4B cells recovered from cellulose acetate filters incubated 7 day in growth chamber
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249) using a quantile normalization (Bolstad et al. Bioinformatics 19(2):185) for RMA background correction and (ii) a median polish summarization to summarize probe data into gene level signal values. The median value was used for the replicate method.
