title: GSM8756656: Wild Type Setaria viridis cv. ME034V-1 - Biological Replicate 1; Setaria viridis; RNA-Seq
library_construction_protocol: To obtain RNA for differential gene expression analyses, leaf discs were taken from plants grown under constant daylight conditions and flash-frozen in liquid nitrogen. Frozen tissue was homogenised using a 1/8” ball bearing with TissueLyser II (Qiagen, Hilden, Germany), shaking at 25 Hz for 1 minute. Total RNA was isolated using a Qiagen RNeasy Plant Mini kit as per the manufacturer protocol. RNA concentration was quantified using a Nanodrop ND-1000 Spectrophotometer at 260 nm and confirmed to be > 280 ng μL-1 for all samples. RNA integrity score was checked using a LabChip GXII Touch (PerkinElmer, Waltham, MA, USA) and confirmed to be > 8.5 for all samples. PolyA-enriched mRNA libraries were prepared using an Illumina TruSeq Stranded mRNA library prep kit following the manufacturer protocol. 1.8 μg of total RNA was reverse transcribed into cDNA for each sample and indexed with IDT for Illumina's Unique Dual Index adapters. cDNA clean-up was performed using Sera-mag SpeedBeads (GE Healthcare, Chicago, IL, USA). cDNA library concentration was determined for each sample using a Qubit High Sensitivity dsDNA Quantitation kit. cDNA library quality was confirmed using a LabChip GXII Touch. Individual library molarity was determined using cDNA concentration and peak fragment size, then normalised to 2.5 nM.  Illumina Next Gen Sequencing was performed at the ACRF Biomolecular Research Facility (The Australian National University, Canberra, ACT, Australia) on the NovaSeq6000 platform (150 bp paired-end sequencing).