title: GSM4880350: WS Root, Flight, Virgin Galactic, replicate 3; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: RNALater™-fixed samples were removed from -80 °C storage, and thawed overnight at 4°C. Seedlings were disentangled, and roots were dissected from shoots. Root tissues were used for RNA extraction, and shoots were restored to -80 °C storage. For each experimental condition, 3-4 biological replicates, derived from roots fixed in a single KFT, were used. Excess RNALater™ was removed from roots, and RNA was extracted using the QIAshredder and RNAeasy kits from QIAGEN (QIAGEN Sciences, MD, USA), using the manufacturer's instructions. RNase-free DNase (QIAGEN GmbH, Hilden, Germany) was used for on-column digestion and removal of DNA. The BO and VG RNA extracts were submitted for RNA-Seq analysis. The total RNA concentration was determined with a Qubit® 2.0 Fluorometer (ThermoFisher Scientific, Grand Island, NY, USA), and RNA quality was assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RINs of the total RNA used for RNA-Seq library construction were between 7.1 and 9.4. RNA-Seq libraries were constructed at the UF ICBR Gene Expression Core using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) following the manufacturer's recommendations. Two ERCC controls were used for the experiment: ERCC Mix 1 was used for flight samples and ERCC 2 was used for ground control samples. Briefly, 1 µl of 1:2000 diluted RNA spike-in ERCC (half the amount suggested in the ERCC user guide) spike to 50 ng of total RNA followed by mRNA isolation using the NEBNext Poly(A) mRNA Magnetic Isolation module (New England Biolabs, Catalog # E7490). RNA-Seq libraries were then constructed with the NEBNext Ultra Directional Library Prep Kit (New England Biolabs, catalog #E7420) according to the manufacturer's user guide. Briefly, RNA was fragmented in NEBNext First Strand Synthesis Buffer by heating at 94 °C for the desired time. This step was followed by first strand cDNA synthesis using reverse transcriptase and oligo(dT) primers. Synthesis of double-stranded cDNA was performed using the 2nd strand master mix provided in the kit, followed by end-repair and Illumina adaptor ligation. Finally, the uniquely-barcoded libraries were enriched by 10 cycles of PCR amplification, and purified by Agencourt AMPure beads (Beckman Coulter, catalog # A63881). 24 barcoded libraries were sized on the Bioanalyzer, and quantitated via the Qubit 2.0 Fluorometer. Finally, the 24 individual libraries were pooled equimolarly.