title: GSM5290488: TTseq_MM1S_2hours_DMSO; Homo sapiens; OTHER
library_construction_protocol: Cells were lysed in in TRIzol. Chloroform (1/5th volume) was added and samples were vortexed and centrifuged at 4°C (13000g, 15 minutes). The aqueous phase was isolated and total RNA was extracted by isopropanol precipitation at room temperature (10 minutes) followed by centrifugation at 4°C (13000g, 10 minutes). The isolated RNA pellet was washed with 70 percent ethanol followed by centrifugation at 4°C (13000g,15 minutes). The RNA pellet was dissolved in 100μL nuclease-free H2O and denatured at 65ºC for 10 minutes. 150μg total RNA per treatment condition (pl 883 us 15μg D.melanogaster S2 RNA spike-in) in 100μL final volume were sonicated in microTUBE AFA Fiber screw cap tubes (6 x 16mm, Covaris, 520096) using the Covaris S220 Focused Ultrasonicator at maximum power for 15 seconds. Thiol-specific biotinylation of fragmented RNA was performed by adding 750μL volume nuclease free water followed by incubatation with 10mM TRIS (pH 7.4), 1mM EDTA, 200 μg/mL dimethylformamide (DMF; Sigma Aldrich, 227056) and 300μg EZ-LinkTM HPDP-Biotin (ThermoFisher Scientific, 21341) at room temperature for 1.5 hours with constant rotation (1500μL final volume – 'biotin labelling reaction'). One volume chloroform was added to the biotin-labelling reaction and the reaction was vortexed and centrifuged at 4°C (20000g, 5 minutes). The aqueous phase was isolated and the chloroform isolation was repeated twice. NaCl (to 0.5M) and one volume isopropanol was added to the aqueous phase and fragmented RNA was isolated by centrifugation at 4°C (20000g, 20 minutes). The RNA pellet was washed with 75 percent ethanol, centrifuged at 4°C (20000g, 20 minutes) and dissolved in nuclease-free water (1μL per 1μg input RNA). Fragmented RNA was denatured at 65°C for 10 minutes prior to incubation with streptavidin beads (μMACs Streptavidin Kit, Miltenyi Biotec, Bergisch Gladbach Germany, 130-074-101) at room temperature for 15 minutes with constant rotation. Biotinylated RNA was isolated using μMACs columns that had been pre-equillibrated with room temperature wash buffer (100mM Tris-HCl pH 7.4, 10mM EDTA, 1M NaCl, 0.1% v/v Tween20). Following incubation with biotinylated RNA / streptavidin beads the columns were washed with 5 times each with 0.9mL pre-warmed (65ºC) and room-temperature wash buffer. Biotinylated RNA was eluted through two additions of 100μL 100mM dithiothreitol (DTT) 3 minutes apart and collected into 700μL RLT buffer from the RNeasy MinElute Cleanup Kit (Qiagen, Hilden Germany, 74204). Biotinylated RNA was isolated using the Qiagen RNeasy MinElute Cleanup Kit using 12μL elution buffer to elute RNA. RNA was quantified using the Agilent Tapestation High Sensitivity RNA kit (Agilent, 5067-5576). Sequencing libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina.