Nuclear-cytoplasmic fractioning performed as described in Wang et al. 2011, RNA purification was performed using TRI Reagent (Sigma)
Label
Cy3
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 in a 65°C oven overnight. Afterwards, the slide were washed in agilent wash buffer 1 and wash buffer2, 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.
