GSM6483005: After-Ripened Dormant Embryo BioRep2; Triticum aestivum; RNA-Seq
Total RNA was extracted from dissected embryos of freshly harvested and after-ripened dormant and non-dormant seeds imbibed for 24 hours using Macherey-Nagel NucleoSpin RNA Plant and Fungi kit (Macherey-Nagel, Düren, Germany). Three biological replicates were used for the analysis. All RNA samples were DNase treated with rDNAse (Macherey-Nagel, Düren, Germany), and subsequently cleaned with NucleoSpin RNA clean-up kit (Macherey-Nagel, Düren, Germany). The purity and concentration of RNA were determined with NanoDrop One spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Agarose gel electrophoresis was used to test RNA degradation and potential contamination. The integrity and quantitation of total RNA was assessed using RNA Nano 6000 Assay Kit on Bioanalyzer 2100 system (Agilent Technologies, CA, USA). The mRNA was enriched using oligo(dT) beads and quantitation to greater accuracy was done by Q-PCR (library activity >2nM). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following standard Illumina platform workflow and protocols with 150 bp paired-end reads.