GSM3790218: Time point 4, replicate 1; Brachypodium distachyon; RNA-Seq
Plants were harvested at 18, 25, 32, and 36 days post sowing. Roots were quickly rinsed, excised from the stem, flash frozen in liquid nitrogen, and stored at -80°C. Frozen root tissue was homogenized in liquid nitrogen using a mortar and pestle. Approximately 100 mg of the resulting frozen root homogenate from each sample was then subjected to RNA isolation using GeneJET Plant RNA Purification Kit (ThermoFisher Scientific, Cat# K0801) according to manufacturer's protocols. The integrity of total RNA was assessed using Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) with the High Sensitivity RNA Analysis Kit. RNA samples without degradation were used for library preparation using the TruSeq Stranded RNA Library Prep Kit (Illumina, San Diego, CA). The sizes of RNA libraries were assessed by Fragment Analyzer with the High Sensitivity NGS Fragment Analysis Kit. The concentrations of RNA libraries were measured by StepOnePlus Real-Time PCR System (ThermoFisher Scientific, San Jose, CA) with the KAPA Library Quantification Kit (Kapabiosystems, Wilmington, MA). The libraries were diluted to 2 nM with RSB (10 mM Tris-HCl, pH8.5) and denatured with 0.1 N NaOH. Eighteen pM libraries were clustered in a high-output flow cell using HiSeq Cluster Kit v4 on a cBot (Illumina). After cluster generation, the flow cell was loaded onto HiSeq 2500 for sequencing using HiSeq SBS kit v4 (Illumina). DNA was sequenced from both ends (paired-end) with a read length of 50 bp. The raw sequencing data BCL files were converted to FASTQ files using software program bcl2fastq2.17.1.14.