Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: For egg cell processing, ovaries were mechanically dissected from emasculated flowers under a dissecting scope, moved to fresh 0.3M mannitol (Sigma) prepared using DEPC-treated water (Sigma) and bisected using a double-edged Merkur (Solingen, Germany) razor blades in 3μL drops of 0.3M mannitol. Using an insect pin, the base of the ovary was gently pushed towards the cut surface until the egg cell became visible. Egg cell protoplasts were then immediately transferred to 1.5mL Eppendorf tubes in liquid nitrogen and stored at -80°C. For sperm cell and vegetative cell cytoplasm sample processing, a differential centrifugation isolation method was used to separate the sperm cells and vegetative cells from whole anthers (Gou et al. 1999, Russell et al. 2012). Briefly, collected anthers were gently ground in cold 45% (w/v) sucrose solution to release mature pollen grains, filtered through 100 μm nylon mesh, resuspended in a 10× volume of 15% (w/v) sucrose solution for 20 min to release the sperm cells, filtered through a 30μm nylon mesh and layered on a Percoll density gradient, centrifuged and recovered as described previously (Russell et al. 2012). The precipitate consisting of vegetative cell materials was treated as a sperm depleted sample and collected in concert with each sperm cell isolation. Therefore, the sperm and vegetative samples represent paired samples, but these were treated as independent when analyzed in conjunction with the egg cell samples. Samples collected were immediately transferred and stored in -80°C until use. RNA from the sperm and vegetative cells was extracted using the QIAGEN RNeasy Plant Mini Kit (QIAGEN) following manufacturer’s instructions including the optional on-column DNase treatment. For the egg cell samples, RNA was extracted using the RNAqueous-Micro Kit (Applied Biosystems). The protocol was modified to add an on-column DNase treatment (QIAGEN) after the first wash step. RNA quality for all samples was assessed and concentrations measured using the Agilent Bioanalyzer (Agilent), and samples had an RNA Integrity Number (RIN) ranging from 7.7 – 9. cDNA synthesis was performed using the NuGEN RNA-seq system V2 (NuGEN) with an input of 1.5 – 10 ng of RNA. cDNA was quantified on the ND 1000 Nanodrop Spectrophotometer (Thermo Fisher Scientific, Inc.) and then sheared on the Covaris S220 system (Covaris) using manufacturer’s recommended settings for 200 bp fragments. 170 ng of sheared cDNA was purified using the MinElute Reaction Cleanup Kit (QIAGEN), and libraries were prepared using the NuGEN Ovation Ultralow DR Multiplex Systems 1-8. Samples were multiplexed and run with 6 samples per lane on the Illumina HiSeq 2000 DNA sequencer (Illumina).