title: HvT3R3
organism: Hordeum vulgare
description: Protocols: The second leaf of the two-week-old seedlings (two leaf stage) were gently brushed 10 times from the base to the top with a soft brush with 7 cm fibres. For RNA-seq transcript analysis, leaves were harvested 0, 25 min, 1h and 2h after touch treatment, and then snap-frozen in liquid N2 and stored at -80°C until use. To study touch-mediated molecular responses in cereal, Triticum aestivum (Chinese spring), Hordeum vulgare (Morex), and Avena sativa (Belinda) were grown as follows. The grains were grown in soil mixture (soil, perlite) under greenhouse conditions (25°C, 16h light/ 8h dark, 100 µmol m-2s-1). For treated samples (time point 22 minutes), the second leaf of the plant was gently brushed around 10 times with a soft hand brush. Data were processed using Demultiplexing and quality controlled with FastQC. Preparation of the samples for RNA isolation was done by grinding them in a mortar and pestle. The mortar and pestle were first cooled using liquid nitrogen and then frozen samples were transferred into the mortar. The samples were ground to a fine powder in the mortar with a pestle while adding liquid nitrogen whenever needed to prevent the sample from thawing. Approximately 100 milligrams of the powdered sample were transferred into microfuge tubes and placed in liquid nitrogen to prevent thawing. Once all 36 samples were prepared, RNA was extracted from them using the Qiagen RNeasy Plant Mini Kit (Qiagen. catalog no: 74904), following the manufacturer's protocol with a slight modification. The DNase treatment was performed to remove genomic DNA contamination, which may produce non-specific amplicons during qPCR reaction. The column was then washed twice with RPE buffer followed by a dry spin for 1 minute to dry the membrane before finally eluting the RNA with 30 µL RNase-free water. 500 ng of total RNA was used for library preparation using Illumina TruSeq mRNA (poly-A selection) and TruSeq RNA UD Indexes for up to 96 samples (Illumina, 20022371).
attributes.tag: replicate
attributes.value: 3
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-04-19
attributes.tag: cultivar
attributes.value: Morex
attributes.tag: organism
attributes.value: Hordeum vulgare
attributes.tag: organism
attributes.value: Hordeum vulgare
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-04-19
attributes.tag: stimulus
attributes.value: brushed
attributes.tag: scientific_name
attributes.value: Hordeum vulgare
attributes.tag: organism part
attributes.value: second leaf
attributes.tag: age
attributes.value: 2
attributes.tag: developmental stage
attributes.value: LP.02 two leaves visible stage
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-04-19
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-04-19