GSM7804600: Sample_CP-B2; Glycine max; RNA-Seq
To assess the effect of rhizobacteria co-inoculation and BPMV on soybean gene expression, we harvested plant leaf tissue from a separate set of untouched undamaged plants at the V4 stage. Tissue sections with weights of 100-200 mg were obtained, and the tissue was flash frozen in liquid nitrogen. The homogenized material was extracted for total RNA using a mirVana miRNA isolation kit (Ambion, Austin, TX). We combined equal amounts of three individual total RNA samples with similar quality and concentration to obtain a pooled replicate sample for each treatment, resulting in a total of five biological pooled replicates per treatment (n = 40). We used a Nanodrop Spectrophotometer (NanoDrop Technologies, Wilmington, DE) to determine RNA concentration and assessed RNA quality using the RNA Integrity Number (RIN) method, which is widely used for RNA quality control (Mueller et al., 2004), in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). To prepare the barcoded libraries for the 40 RNA samples, 100 ng of total RNA was used with a TruSeq Stranded mRNA Library Kit, following the manufacturer's instructions. The concentration of each library was determined by qPCR, and an equimolar pool of the 40 barcoded libraries was created.