title: Sick1
organism: Malus domestica
description: Protocols: The cross 'Braeburn' (susceptible to bitter pit) × 'Cameo' (resistant to bitter pit) (B×C) was performed at Fondazione Edmund Mach (FEM) in 2004. A total of 1,266 seedlings were raised, grafted onto 'M.9' rootstocks and maintained following standard technical management procedures in a field environment at Spini di Gardolo in Trentino, Italy. The entire B×C progeny were phenotyped for bitter pit symptoms over 2 growing seasons. Apple peels from three susceptible and three non-susceptible seedlings were collected at 21 and 147 days post fertilization (full maturation stage) and used for RNA extraction. From each sample, 50 mg of apple peel were used to extract total RNA using the Spectrum™ Plant Total RNA Kit (Sigma-Aldrich) with the addition of an on-column DNAase treatment (Sigma-Aldrich). The extraction method followed the manufacturer's instructions with two modifications: 1% PVP was added to lysis solution, and the number of washings was doubled, meaning two washings with wash solution 1 and four washings with wash solution 2 were performed per extraction. Each extracted RNA was diluted in 50 μl of elution solution. The quality and quantity of the RNA were measured using a NanoDrop spectrophotometer (Thermo Scientific), and an Agilent 2100 BioAnalyzer using the Agilent RNA 6000 Nano Kit. Total RNA samples were suitable for RNA-Seq analysis when their concentration was greater than 50 ng/μl, 260/280 and 260/230 ratios were above 1.8 and 2.0 respectively and when the RIN (RNA integrity number) was above 7.00. A total of four RNA pools were made for RNA-Seq analysis: total RNA from the three susceptible plants and the three resistant plants was pooled from developmental stages 1 (21 days after fertilization) and 7 (147 days after fertilization) to yield: (a) susceptible pool stage 1, (b) susceptible pool stage 7, (c) resistant pool stage 1, and (d) resistant pool stage 7. Each RNA pool was obtained by combining the same quantity of RNA from each extracted sample. A total of 2 μg of RNA per pool was used for RNA-Seq library construction. Libraries were prepared using the TruSeq RNA sample preparation kit (Illumina).
attributes.tag: ENA first public
attributes.value: 2017-12-31
attributes.tag: ENA last update
attributes.value: 2017-11-06
attributes.tag: External Id
attributes.value: SAMEA104369051
attributes.tag: INSDC center alias
attributes.value: BIOGEST-SITEIA, Department of Life Sciences, University of Modena and Reggio Emilia
attributes.tag: INSDC center name
attributes.value: BIOGEST-SITEIA, Department of Life Sciences, University of Modena and Reggio Emilia
attributes.tag: INSDC first public
attributes.value: 2017-12-31T17:01:46Z
attributes.tag: INSDC last update
attributes.value: 2017-11-06T12:29:54Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6190:Sick1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: apple
attributes.tag: cultivar
attributes.value: Braeburn x Cameo
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: pool 2 of 3 mixed samples
attributes.tag: organism part
attributes.value: pericarp
attributes.tag: phenotype
attributes.value: severe bitter pit symptoms
attributes.tag: sample name
attributes.value: E-MTAB-6190:Sick1
attributes.tag: sampling time point
attributes.value: 21 days post anthesis