Instrument: Illumina HiSeq 4000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Samples were harvested at 4.5 G for 10 minutes, resuspended in 1 ml TRI reagent, and incubated at room temperate for 5 minutes. Amylene-stabilised chloroform (400 µl, Sigma-Aldrich 34854-M) was added to each tube, and incubated for 2-15 minutes at room temperature. Samples were then transferred to 5Prime Phase Lock Gel 'Heavy' tubes (VWR 733-2478) and centrifuged at 17 G for 15 minutes at room temperature. The aqueous phase was collected into a fresh microfuge tube and 450 µl isopropanol was added to precipitate RNA. Samples were incubated at room temperature for 30 minutes before pelleting at 17 G for 30 minutes. The pellet was rinsed by gently adding 1 ml of 70% ethanol and pelleting at 7.5 G for 5 minutes. The supernatant was carefully removed and the pellet allowed to air dry before resuspension in RNase-free water at 65°C. To remove any residual contaminating DNA, RNA was diluted to ≤200 ng/µl and treated with TURBO DNA-free RNase-free DNase (Invitrogen AM1907) according to the manufacturer's instructions. Reaction products were purified with Agencourt RNAClean XP beads. Ribosomal RNA was removed using the Bacterial RiboZero rRNA depletion kit (Illumina). NEBNext Ultra-Directional RNA library preparation kit (Illumina). Sequencing is 'reverse-stranded'.