Ribominus rna plant kit for rna seq

Frozen plant samples were ground in liquid nitrogen and total RNA was extracted using One Step RNA Reagent (Bio Basic Inc., Canada) as per the manufacturer's protocol and purified using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA). The integrity of the RNA was assessed by formaldehyde agarose gel electrophoresis. Total RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and a Bioanalyzer 2100 (Agilent Technologies, CA). RNA integrity number (RIN) values were greater than 8.0 for all samples. Ribosomal RNA depletion was carried out using a RiboMinus RNA plant kit for RNA-Seq (Life Technologies, CA). The whole-transcriptome cDNA library was prepared using an Ion Total RNA-Seq kit v2 (Life Technologies Corporation, CA). Double-stranded cDNA was ligated to barcoded adapters and sequenced by BGI-Shenzhen Ltd. (Shenzhen, China) using an Ion PI Chip (ion torrent, Life Technologies, CA). Processing of raw data, removal of adapter sequences, base-calling, and quality value calculations were performed using Torrent Suite Software 4.0 (ion torrent, Life Technologies, CA). Quality reads were obtained by trimming the raw reads at a minimum PHRED score of Q = 20.

Samples with RNA Integrity Score (RIS) values greater than 8.0 were further processed for preparing cDNA library. Ribosomal RNA depletion was carried out using RiboMinus RNA plant kit for RNA-Seq (Life Technologies, CA). The whole transcriptome cDNA library was prepared using Ion Total RNA-Seq kit V2 (Life Technologies Corporation, CA). Double stranded cDNA was ligated to barcoded adapters, loaded onto the Ion PI™ Chip (Ion torrent, Life technologies, CA) and sequenced in triplicate according to the standard protocol using Ion Proton System (Ion torrent, Life technologies, CA).