GSM4661605: VT40-a; Vitis vinifera; RNA-Seq
Total RNA was extracted using a Plant RNA EASYspin Plus Kit (aidlab, Peking, China) according to the instructions of the manufacturer, and treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm and 280 nm (A260 and A280) using SmartSpec Plus (BioRad, USA). RNA integrity was further verified by electrophoresis using a 1.5% agrose gel. To avoid individual variation, we selected three biological replicates for each sample, and extracted total RNA separately, and then we pooled the replicates together for the following experiments. Construction of cDNA library was independently prepared using 10 ug of total RNA. Polyadenylated RNAs were purified and concentrated with oligo (dT)-conjugated magnetic beads (Invitrogen) before being used for the directional RNA-seq library preparation. Purified mRNAs were iron fragmented at 95oC followed by end repair and 5' adaptor ligation. Then reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified and amplified and PCR products corresponding to 200-500 bps were purified, quantified and stored at -80 oC until used for sequencing.
GEO Accession
GSM4661605