description: Protocols: Sampling of healthy and botrytized Vitis vinifera cv. Furmint berry samples was performed in Mád (Tokaj wine region), Hungary in vineyard Betsek, 48°11'16\N 21°19'03\E, on October 25 and November 3, 2016. Four different types of berry samples were collected starting with healthy berries followed by 3 consecutive stages of noble rot. Stage I. was characterized by partly botrytized epidermis and no shrivelling, stage II. berries showed fully colored epidermis and mild shrivelling while stage III. exhibited all features of mature noble rot berries (fully shrivelled with only limited presence of B. cinerea material on surface). Both sampling times five biological replicates were collected in 50 ml centrifuge tubes for all four stages of noble rot, one sample comprising 5 (healthy) to 15 (stage III.) berries. Samples were frozen immediately in liquid nitrogen, transported in dry ice and stored at -70 °C in an ultra low temperature laboratory freezer. RNA-Seq data were generated from samples collected within the frame of a field experiment. Description of the vineyard and viticultural practices have been described by Hegyi-Kaló et al. (2020). Reference: Hegyi-Kaló J., Hegyi Á.I., Geml J., Zsófi Z., Pálfi X., Váczy, K. Z. (2020) Physico-chemical characteristics and culturable microbial communities of grape berries change strongly during noble rot development. Plants 9: 1809. Berries were ground to a fine powder in liquid nitrogen by a Retsch laboratory ball mill. Considering the uneven dry weight of the berry samples varying amounts of frozen plant powder were weighed into new 50 ml centrifuge tubes, 800 mg for healthy and stage I. berries, 500 mg for stage II. berries and 250 mg for stage III. berries. Total RNA extraction was carried out according to Reid et al. (2006). Twenty milliliter extraction buffer [300 mM Tris HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, 2% CTAB, 2% PVPP, 0.05% spermidine trihydrochloride, 2% β-mercaptoethanol (administered immediately before use)] was added to powdered samples incubated at 65 °C for 10 minutes and vortexed every 2 minutes. After that, mixtures were extracted twice with equal volumes of chloroform:isoamyl alcohol (24:1), thoroughly vortexed and centrifuged at 4800 x g for 10 minutes at 4 °C. The supernatant (10 ml) was transferred to a new 50 ml centrifuge tube and combined with 0.1 vol 3M NaOAc (pH 5.2) and 0.6 vol ice-cold isopropanol, mixed and kept for 30 minutes at -70 °C. The nucleic acid pellets were recovered by centrifugation at 4800 x g for 30 minutes at 4 °C. The supernatant was removed and the remaining pellet was washed with 5 ml of 70 % EtOH, then centrifuged at 4800 x g for 15 minutes at 4 °C. The liquid was discarded and the precipitate was dried with a lyophilizer.The nucleic acid pellets were dissolved in 1 ml TE (pH 7.0) and transferred to new 2 ml microcentrifuge tubes. Selective RNA precipitation was achieved by adding 0.3 vol of 8M LiCl and a subsequent overnight incubation at 4 °C. Total grapevine and fungal RNA were harvested by centrifugation at 21000 x g for 30 minutes at 4 °C followed by a washing step with 500 l 70 % ice-cold EtOH and finally drying by a lyophilizer. Isolated RNA pellets were dissolved in 42 microliter nuclease-free ultrapure water and RNA quality was first evaluated by using a nanodrop 1000 instrument. After the extraction was finished all samples were subjected to DNase treatment (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, DNA-free™ DNA Removal Kit). Total RNA sample quality was analyzed more precisely on an Agilent BioAnalyzer using Eukaryotic Total RNA Nano and Pico Kit according to the manufacturer's protocol. Samples with RNA integrity number (RIN) value >7 were accepted for library preparation process.Cited literature: Reid K.E., Olsson N., Schlosser J., Peng F., Lund S.T. (2006) An optimized grapevineRNA isolation procedure and statistical determination of reference genes for real-timeRT-PCR during berry development. BMC Plant Biology 6:27. RNA-Seq libraries were prepared from total RNA using TruSeq RNA Sample preparation kit (Illumina) according to the manufacturer's instructions. Shortly, poly-A RNAs were captured by oligo-dT conjugated magnetic beads then the eluted mRNAs were fragmented at 94 °C. First strand cDNA was synthesized by random priming reverse transcription and after second strand synthesis step double stranded cDNA were produced. Following repairing ends, A-tailing and adapter ligation steps adapter ligated fragments were amplified in enrichment PCR and lastly double stranded mRNA sequencing library was generated.
attributes.tag: ENA first public
attributes.value: 2021-11-25
attributes.tag: ENA last update
attributes.value: 2021-11-25
attributes.tag: External Id
attributes.value: SAMEA11296459
attributes.tag: INSDC center alias
attributes.value: Agricultural Institute, Centre for Agricultural Research, ELKH
attributes.tag: INSDC center name
attributes.value: Agricultural Institute, Centre for Agricultural Research, ELKH
attributes.tag: INSDC first public
attributes.value: 2021-11-25T00:18:35Z
attributes.tag: INSDC last update
attributes.value: 2021-11-25T00:18:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-11205:CFIV-3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: wine grape
attributes.tag: cultivar
attributes.value: Furmint
attributes.tag: disease
attributes.value: stage III. Noble rot
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: growth condition
attributes.value: field study
attributes.tag: organism part
attributes.value: berry
attributes.tag: phenotype
attributes.value: mature noble rot berries, fully shrivelled
attributes.tag: replicate
attributes.value: 3/5
attributes.tag: sample name
attributes.value: E-MTAB-11205:CFIV-3
attributes.tag: sampling time point
attributes.value: October 25, 2016