title: RNAseq of Arabidopsis thaliana central cell before fertilization
design: Pistils obtained from crosses were collected at precise time points, fixed in ice-cold 9:1 (v/v) ethanol:acetic acid o/n at 4C, then transferred in 70% ethanol and moved to an ASP200 embedding machine (Leica Microsystems GmbH, Wetzlar, Germany), where the tissue was dehydrated in a graded series of ethanol at RT (70% for 1 h, 3 90% for 1h, 3 99.98% for 1h), followed by xylol (2 for 1 h, 1 for 1 h 15 min). Xylol was substituted by Paraplast X-tra embedding media (Roth AG, Arlesheim, Switzerland) at 58C (2 for 1 h, 1 for 3 h). The pistils were poured into small plastic trays while the paraffin was still liquid and let to set. For microdissection, paraffin blocks containing the pistils were cut on a RM2145 Leica microtome (Leica Microsystems GmbH, Wetzlar, Germany) to 8 m-thin slices and mounted on nuclease-free membrane-mounted metal-frame slides (MicroDissect GmbH) using DEPC water. The slides were then submerged at RT in xylol to remove the paraffin, with two washes of 20 min each. Microdissection was performed using a MMI CellCut Plus device (MMI Molecular Machines & Industries AG, Glattbrugg, Switzerland). After collection, 50 l of extraction buffer of the PicoPure RNA isolation kit (Arcturus Engineering, Mountain View, USA) was added, the sample incubated for 30 min at 42C and then stored at 80C. Total RNA was isolated using the PicoPure RNA isolation kit (Arcturus Engineering, Mountain View, USA) following the manufacturer's instructions. Total RNA was tested for integrity and quality using the Agilent TAPE Station and was then converted to cDNA using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio) following the manufacturers instructions using OligodT. Libraries were constructed using the Nextera XT DNA Sample Preparation Kit (Illumina) following manufacturers instructions. Libraries were then sequenced at 150bp double paired-end, in a Nova-Seq Illumina machine, by combining all 12 libraries in a single lane at the Functional Genomic Center Zurich.