GSM5387735: Control_1h_A; Setaria viridis; RNA-Seq
For each biolgoical replicate, 2cm mid-leaf segments from two plants for each time point and treatment were collected from 4th fully expanded true leaves into screw cap tubes with grinding bead and immediately frozen into liquid nitrogen and stored at -80oC.  Frozen samples were homogonized using paint shaker.  RNA was extracted using Trizol method with all centrifugation at 4oC and 11,000 RCT.  1ml of Trizol reagent was added to homogenized leaf tissue and resuspended, then 200uL of Chloroform:Isoamyl alcohol (25:1) was added and vortexed.  Tubes were centrifuged for 15 min. 6-- uL from the aqueous layer was transferred to a clean tube with equal volume Chloroform:Osoamyl alcohol, vortexed, and centrifuged for 5 min. 450uL of aqueous layer was transferred to 0.7x volume 100% Isoporopanol, mixed well, and chilled for 30 min at -20oC. Samles were centrifuged for 15 min to pellet RNA.  Supernatent was decanted and RNA pelled was rinsed twice with ice-cold 75% ethanol with a 2-min centrifiugation following each rinse. RNA was dried in a laminar flow hood until residual ethanol evaporated and was resuspended in 50uL nuclease free H2O. RNA was quantified using NanoDrop and Qubit RNA Broad Range Assay Kit. RNA integrity was verified using a Bioanalyzer Nano Assay. RNA samples were diluted to 200 ng/uL in nuclease free H2O for a total of 1 uG RNA.  Libraries were generated with the Quantseq 3' mRNA-seq library prep kit FWD for Illumina. Libraries were generated according to manufacturer's instruction. Cycle count for library amplification for 1 ug mRNA was tested using the PCR add-on kit for Illumina. qPCR was performed and a cycle count of 13 was determined for the amplification of all libraries. For library amplification, the Lexogen i5 6nt Dual Indexing Add-on Kit was used in addition to the standard kit to allow libraries to have a unique combination of i5 and i7 indices.  All libararies were quantified using Qubit dsDNA High Sensitivity Assay Kit with the Qubit 3.0 machine.  Prepared libararies were pooled to equimolar concentrations based on Qubit assay reads.  Poolec libaries were submitted to Novogene to be sequenced on the Illumina HiSeq 4000 platform.