GSM2337102: PBS1WKS1; Vitis vinifera; RNA-Seq
Total RNA was extracted from 0.1 g of ground tissue per sample. The frozen tissue was homogenized by inversion in 500 µl of extraction buffer (PureLink Plant RNA Reagent, Ambion cat# 12322-012) for 5 min at room temperature. After homogenization, 100µl of Plant RNA isolation Aid (Ambion cat# AM9690) was added to the suspension and mixed by inversion for 5 min at room temperature. Samples were centrifuged at top speed for 5 min at room temperature to pellet insoluble debris and other contaminants. The supernatant was transferred to a new tube and then 100µl of 5M NaCl and 300µl chloroform were added and mixed for 5 min by inversion. Samples were centrifuged at 12,000 xg for 10 min at 4oC to separate the phases, and the upper aqueous phase was transferred to a new tube. Samples were treated with DNase RNase-free (5 U) and incubated for 20 min at 37oC. After DNase treatment, 500µl of chloroform was added and mixed for 5 min as described above. To separate the phases, the samples were centrifuged again at 12,000 xg for 10 min at 4oC, and the upper aqueous phase was transferred to a new tube. An equal volume of isopropyl alcohol was added, after the samples were mixed by inversion and incubated at room temperature for 10 min. Precipitated RNA was centrifuged at 12,000 xg for 10 min at 4oC, and the supernatant was discarded. The RNA pellets were washed with 1mL of 80 % ethanol and centrifuged at 12,000 xg for 2 min at room temperature. Then the pellets were air-dried at room temperature and re-suspended in 30µl RNase-free water. Illumina TruSeq RNA Sample Prep Kit v2 protocol
GEO Accession
GSM2337102