tissus: seed genetic background: A17 developmental stage: 28_DAP maturation phenotype: Wild-type treatment: Seeds developed at 15-13°C 16h age: 28 days after flowering harvest date: 06/06/12
Treatment protocol
Medicago truncatula seeds developed at 14/11°C, 16h light/darkf rom 1 DAP to DS stage
Growth protocol
Five Medicago truncatula plants were grown at controlled light/temperature conditions: 14/11°C, 16h light/dark from the stage of flowering to the end of seed maturation. Seeds were collected at different maturation stages, from 22 DAP to Abscission. Dry seeds (DS) was the last analysed stage of development, after abscission seeds were let in the same environmental conditions for one week.
Extracted molecule
total RNA
Extraction protocol
For each data point of seed development and for each repetition, 50 seeds were collected and the total RNA was extracted with Macherey-Nagel's Nucleospin Plant II Kit according manufacturer's recommendations
Label
Cy5
Label protocol
400ng of total RNAs were amplified using the Ambion messageAmp II (Ambion, Austin TX) following manufacturer's instructions. 5µg of each aRNAs were retrotranscribed with 400U of Superscript II reverse-transcriptase (Invitrogen Corp., Carlsbad, CA) and labelled with 1,5mmol of Cyanine-3 (Cy3) or Cyanine-5 (Cy5) (Interchim, France), then purified with NucleoSpin Gel and PCR Clean-up column kits (Macherey-Nagel, GmbH & Co. KG, Germany). Purified labeled cDNA were quantified using NanoDrop ND-1000 (Nanodrop Technologies, DE, USA). Labelled samples were combined as 30 pmol for each dye and co-hybridized to the Medtr_v1 12x135K arrays.
Hybridization protocol
The hybridization performed on a NimbleGen Hybridization System 4 (mix mode B) at 42° overnigth. Afterwards, the slide were washed, dried, and scanned.
Scan protocol
Slides were scanned at 532/635nm at 2 μm resolution and high sensitivity with a Roche-NimbleGen MS200. NimbleScan Software v2.4 was used to extract pair-data files from the scanned images.
Data processing
All statistical analyses on the gene expression data were performed using the R language, version 2.5.1 (R Development Core Team, 2011) and the package LIMMA (Smyth G.K., 2005) from the Bioconductor project. For the processing step, data were normalized by the lowess method in LIMMA.
