title: GSM5795759: sparse panicle1 mutant inflorescence_14DAS_rep2; Setaria viridis; RNA-Seq
library_construction_protocol: Inflorescences were hand-dissected into fresh 100% acetone on dry ice from wildtype (A10.1) and mutant (sparse panicle1) S. viridis seedlings at 10, 12, and 14DAS. Depending on the representative size at a given stage, 10-30 individual inflorescence primordia were pooled per biological replicate; three to four biological replicates were collected for each stage. All sampling was performed within a two-hour window in the morning to control for circadian effects. Acetone was removed and samples were flash-frozen in liquid nitrogen and ground into a fine powder using 3mm tungsten-carbide beads (Qiagen) in a Tissue Lyser-II. Total RNA was isolated using the PicoPure RNA Isolation Kit (Thermo Fisher Scientific) with in-column DNase I treatment following manufacturer's protocols. RNA-seq libraries were generated from 1µg total RNA using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs Inc.) and size-selected for 200 bp inserts. Libraries were quantified on a bioanalyzer (Agilent 2100) using a DNA 1000 chip to confirm the insert size and quantified again on a Qubit 3.0 Fluorometer (Life Technologies) to ensure different libraries were equally loaded Libraries were sequenced using standard Illumina protocols (Illumina, Inc.) for either paired-end (PE) sequencing at 100bp on the Illumina HiSeq 2500 platform at the University of Illinois at Urbana-Champaign W.M. Keck Center.