cultivar: Cigalon tissue: root days post inoculation: 7
Growth protocol
Before inoculation, Azospirillum 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. Rice seed sterilization was performed as previously described by Drogue et al. 2014 (FEMS Microbiol. Ecol.). Four plant were added in each square plate. 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 experiment, 30 plant root systems were pooled and frozen using liquid nitrogen. Root cell lysis was performed by grinding root systems with a mortar and pestle under liquid nitrogen. Total RNA was isolated using the TRIzol method (Invitrogen, Carlsbad, CA, USA). RNA samples were purified using RNeasy plant mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s protocol. RNA integrity was assessed using Agilent RNA 6000 Pico Kit (Agilent Technologies, Waldbronn, Germany) and the Agilent 2100 Bioanalyzer (Agilent Technologies) device. In order to increase mRNA representation in RNA samples, total RNA were digested with mRNA ONLYTM Procaryotic mRNA isolation kit (Epicentre Biotechnologies, Madison, WI, USA) according to the provided protocol. The microarray cDNA (three independent samples per condition) was synthesized with the SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen), using a mix (1:1) of random primers (Promega Corporation, Madison, WI, USA) and Oligo-dT(15) primers (Promega).
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
Scanning was performed by NimbleGen Systems Inc., Madison, WI USA, following their standard operating protocol. See www.nimblegen.com.
Description
This sample is the second replicate of non-inoculated roots of Oriza sativa japonica cv. Cigalon
Data processing
The raw data (.pair file) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185) Intensity values were summarized using median polish algorithm. The median value was used for the replicate method.
