Ovules were collected from ~60 siliques using vacuum extraction. Siliques were stuck to double-sided tape and sliced open using a needle. Open siliques were submerged in 1x PhosphateBuffered Saline (PBS) buffer and ovules were collected using a vacuum pump through 50 µm filters. Collected ovules were then transferred to Isolation buffer (1x First Strand Buffer (FSB; Invitrogen), 1mM Dithiotreitol (DTT), 4% RNAseLater, MQ), and volume was reduced to ~20 µL. Embryo isolation was performed according to (Raissig, 2013) with the following adaptations. A Zeiss Confocor 1 inverted microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) together with an Eppendorf Transferman 4r micromanipulator (Eppendorf AG) and VacuTip II microcapillaries (Eppendorf) were used to isolate about 40-50 washed embryos in 50 μl isolation buffer.
Growth protocol
All seeds were sterilized in 25% bleach/75% ethanol solution for 10 minutes and were afterwards washed twice with 70% ethanol and once with 100% ethanol. Dried seed were plated on half-strength Murashige and Skoog (MS) medium and the appropriate antibiotic (in concentration: 50 mg/l kanamycin or 15 mg/l phosphinothricin) for selection of transgenic seeds. After 24hours incubation at 4°C, the plants were grown under long-day conditions at 22°C in a growth room.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Trizol-Chloroform extraction followed by clean-up using the RNeasy Plant Mini kit from Qiagen. RNA integrity was checked on chip analysis (Agilent 2100 Bioanalyzer, Agilent Technologies, Amsterdam, The Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label
biotin
Label protocol
One hundred nanogram of RNA was used for whole transcript cDNA synthesis with the Ambion WT expression kit [catalog number 4411974] (Applied Biosystems/Life Technologies, Nieuwekerk a/d IJssel, The Netherlands).
Hybridization protocol
Hybridization of 5.5μg labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridization Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Washing and staining of the arrays were done on an Affymetrix 450 Fluidics Station using the protocolFS450_0002, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5).
Scan protocol
Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the GeneChip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
Data processing
Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'affy' (v1.44).
