GSM7851098: 9DPA_32C_biorep2; Solanum lycopersicum; RNA-Seq
Nuclei were prepared from 20 tomato fruits pericarp at 6DPA and 10 fruits at 9DPA and 12DPA. Nuclei were sorted by FANS as described in (Bourge et al., 2018). Pericarp pieces were chopped using a razor blade in 2mL of Gif Nuclear Buffer (GNB: 45 mM MgCl2, 30 mM Sodium-Citrate and 60 mM MOPS pH 7.0, 1% PVP 10.000, 0.1% Triton X-100 and 10 mM sodium metabisulfite (S2O5Na2)) and the suspension was filtered twice through a 48 µm nylon mesh before adding DAPI (4',6-diamidino-2-phenylindole). 100k nuclei were sorted according to their ploidy levels with a MoFlo ASTRIOS EQ (BECKMAN COULTER) and collected in a tube containing 900µL of TRIzol™ Reagent (THERMOFISHER SCIENTIFIC). Before RNA extraction, 1µL of a dilution 1:1000 of ERCC RNA Spike-In Mix (THERMOFISHER SCIENTIFIC) was added. The mix of nuclei and spike-ins were shortly vortexed and rested for 5mn at room temperature before adding 200µL of Chloroform, agitating the tubes by hand for 15sec and incubating them for 3min at room temperature. After the incubation, tubes were centrifuged for 15mn at 12000g and 4°C, and the upper aqueous phase was transferred (around 840µL) to a fresh tube and mixed with 420µL of 100% ethanol. Then, total RNA was further purified using the RNeasy Micro Kit (QIAGEN) according to the provider's instructions. A final volume of 14µL of total RNA was obtained. Total RNA was used for construction of libraries following the instructions from the Illumina TruSeq Stranded RNA Sample Prep Kit. After library preparation for sequencing, a Duplex-specific nuclease (DSN) treatment was done using the commercial enzyme from EVROGEN following manufacturer's instructions. Following DSN treatment, libraries were purified with SPRI beads (BECKMAN) and paired-end sequenced (2x150bp) with a HiSeq3000 (ILLUMINA).