At ten stages seeds were disected using a scalpel and a dissecting needle into the above specified compartments
Growth protocol
Seeds of Lepidium sativum FR14 (Quedlinburger Saatgut GmbH) were sown on filter disks with 6ml of sterile, pure water and incubated at 24°C with continuous light
Extracted molecule
total RNA
Extraction protocol
Lepidium seed tissues (100 radicles or 100 micropylar endosperms, 100 non-micropylar endosperms, 50 cotyledons) were homogenized twice in liquid nitrogen with the precellys homogenisator at 6100 rpm (peqlab, Erlangen), thawed in 1 ml CTAB-buffer (2% [w/v] hexadecyl trimethyl-ammonium bromide (CTAB); 2% [w/v] polyvinylpyrrolidone PVP (MW=40000/K30); 100 mM Tris-HCl, pH 8.0; 25 mM EDTA, pH 8.0; 2 M NaCl; 2% [v/v] ß-mercaptoethanol) homogenized again and then incubated at 65°C for 15 min. After two extraction steps with 1 ml chloroform:isoamylalcohol (24:1 [v/v]) the volume of the hydrophilic phase was determined and ¼ of that volume 10 M LiCl was added. The RNA was precipitated at 4°C overnight. After centrifugation at 13.000 rpm and 4°C, the pellet was resuspended in 600 μl SSTE-buffer (1 M NaCl; 0.5% [w/v]sodium dodecyl sulfate (SDS); 10 mM Tris-HCl, pH 8.0; 1 mM EDTA) and two more chloroform extractions were performed in PhaseLock vials (5prime, Hamburg). The RNA in the hydrophilic phase was precipitated with Na-Acetate/Ethanol at -20°C, the pellet washed with Ethanol, air-dried and resuspended in RNAse-free water. An RNeasy column clean-up was performed according to the manufacturer’s instructions to remove remaining impurities (Qiagen, Hilden). The optional DNAse treatment was performed on the column according to manufacturer’s instructions.
Label
biotin
Label protocol
The Affymetrix 3' IVT-Express Labeling Kit was used to synthesize Biotin-labeled cRNA. For each sample 100 ng was used as input.
Hybridization protocol
Fragmented cRNA at a final concentration of 0.0375ng/μl was used for hybridization. The GeneChip (#900720) HWS Kit was used for hybridization, washing and staining.
Scan protocol
The scanning was performed using the Affymetrix 3000G scanner
Data processing
The data was analysed using RMA in the affy Bioconductor package in R, using the v14.0.0 Arabidopsis CustomCDF from http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF along with a novel cross-species masking technique involving taking an ANOVA for each probe to determine whether it is registering signal or noise. See paper for details.
