RNA-seq analysis of grafting and wounding grapevine stems
Dormant, overwintering one year old stems of grapevine were harvested from the vineyard and stored at 4 °C until use. Scions, rootstocks and cuttings were soaked in water at room temperature for 4 h one day before grafting/wounding. Internodes were sampled just before grafting/wounding treatments from the three genotypes studied (time point 0).  For each graft combination, grafts were made using an Omega Star grafting machine (Chauvin, France), and wounded and intact cuttings were made (both with and without buds removed). Both grafts and cuttings were dipped in melted wax (Staehler Rebwachs pro with 0.0035% of dichlorobenzoic acid, Chauvin). The grafts/cuttings were then placed in plastic boxes for 5 d at room temperature and then placed in a room at 28 °C with high humidity and with 2 cm of water at the bottom of each box. The grafts and, intact and wounded cuttings, with or without buds, were sampled at 14 d after wounding/grafting. For this, four pools of five grafts/cuttings were randomly sampled. Approximately 0.5 cm of tissue was taken from above or below the graft interface or wound site, and immediately immersed in liquid nitrogen and stored in the freezer at -80°C. For the cuttings without wounds, 1 cm of wood was sampled at the same location as the graft interface or wound site.  Finally, samples were ground in a ball mill (MM400 RETSCH) in liquid nitrogen at 30 Hz during 30 s and directly kept at -80°C waiting for future analysis.  One year old grapevine stems were purchased from the Chambre de l'Agriculture de l'Aude (Carcassonne, France) for Vitis vinifera cv. Pinot noir and V. berlandieri x V. rupestris cv. 140 Ruggeri, and from Mercier (Vix , France) for V. riparia cv.  Gloire de Montpellier. Total RNA was extracted from samples using Spectrum Plant Total RNA Kit (Sigma-Aldrich) following the instructions with some modifications. 80-100 mg of powder was used for the extraction. For each sample, 900 µL of lysis solution was used with 0.9 µL of β-mercaptoethanol and 40 mg of polyvinylpolypyrrolidone (PVPP). After adding the modified lysis buffer to the samples, 500 µL of a chloroform:IAA (24:1) mixture was added and tubes were centrifugated at 13200 rpm during 5 mins. After collecting 650 µL of supernatant and adding 650 µL of ethanol, the following steps corresponded to the protocol provided in the extraction kit. At the end, RNA was eluted from 50 µL of elution solution and kept in the fridge at -80°C. Libraries were generated at BGI according to the DNBSEQ stranded mRNA library system.