Ion total rnaseq v2 kit

As reported previously [11 (link)], RNA extraction was performed using the Total Exosome RNA and Protein Isolation Kit (catalog # 4478545; Invitrogen, USA) according to the provided instructions. 200 ng-1 μg RNA in final volume of 30 μL solution was collected for each sample. Total RNA quantity and quality (260/280 absorbance ratio) were assessed using NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer to test concentration and inorganic ions or polycarbonate contamination. miRNA sequence was isolated by BGI Company (China) based on previous instructions [12 (link)]. cDNA libraries were constructed using the Ion Total RNA-Seqv2 kit (Life Technologies, USA) (n = 3 for each group) and purified using AMPure beads (Beckman Coulter). Emulsion PCR and enrichment of cDNA-conjugated particles were performed with an Ion OneTouch 200 Template Kit v2 DL (Life Technologies). The final cDNA samples were sequenced single end on the HiSeq 2000 System with a 50 bp read length.

Saccular RNA samples from all three sexual phenotypes (type I male, type II male, reproductive female) were sent to the Laboratory of Biotechnology and Bioanalysis at WSU Pullman for Ion Torrent sequencing. One nanogram of mRNA was used to construct RNA-seq libraries using the Ion Total RNA Seq V2 kit (Life Technologies) low input protocol, with the exception that AMPureXP SPRI beads (Beckmann Coulter) were used for all purifications during library construction. Final library size selection was achieved with 0.7X AMpureXP. The libraries were quantified by qPCR and sequenced separately on an Ion Torrent PGM using a single Ion 318 chip for each sample and sequencing beads produced on an Ion OneTouchDL using 200bp chemistry.

For more details, see [14 (link)]. Briefly, a population of P. vulgaris plants were grown under greenhouse conditions. Floral buds (excluding sepals) were collected from both mating types at two developmental time points (3–4 mm and 10+ mm bud sizes), flash frozen, and stored at −80 °C. For each sample, 4 g of floral buds from six independent plants were used for RNA extraction and subjected to mRNA purification prior to library construction. Sequencing libraries for each mRNA sample were constructed from 200 ng of polyA mRNA using the Rapid RNA Library Kit (Roche, Basel, Germany), and sequenced with a Roche 454 FLX titanium (Roche, Basel, Germany). To increase sequencing depth in the young bud samples, mRNA from 3–4 mm thrum and 3–4 mm pin samples was also used to generate Ion Torrent sequencing libraries using the Ion Total RNA Seq V.2 kit (Life Technologies, Carlsbad, CA, USA), and sequenced with an Ion Torrent PGM. All library construction and sequencing was performed by the Washington State University Genomics Core Laboratory. Sequencing reads were trimmed to remove adapters, quality filtered using default parameters, and then used for de novo transcriptome assembly. Assembly was performed using the SeqMan NGen assembler (DNAStar, Madison, WI, USA), with default parameters (match size 21, match spacing 75, minimum match percentage 85%).