title: GSM5384970: 20-minute TT-seq 2 lane2; Mus musculus; OTHER
library_construction_protocol: Cells were counted and collected in Trizol, to which was added 5% S2 cells, labelled with 4sU for 2 hours. RNA was chloroform extracted, DNase-treated, and chloroform extracted. To 60 μg total RNA was added 2X fragmentation buffer (final concentration: 75 mM Tris Cl, pH 8.3, 112.5 mM KCl, and 4.5 mM MgCl2) and heated to 95 C for 5 minutes. Fragmentation was stopped by adding EDTA to 50 mM and placing samples on ice. RNA was ethanol precipitated and resuspended in H2O. Fragment sizes were checked on the TapeStation (peak size of ~800 nt). Biotinylation reaction was performed with 0.025 mg/mL MTSEA-Biotin XX (Biotum) in reaction buffer (20% N,N-Dimethylformamide, 1mM EDTA, 10 mM HEPES pH 8) for 45 minutes in the dark. Labelled RNA was chloroform extracted and ethanol precipitated, and resuspended in 90 μL H2O. 75 μL DynabeadsTM M-280 Streptavidin (ThermoFisher) were pre-washed with decon solution (0.1M NaOH + 50mM NaCl), 2X 100mM NaCl, 2X High Salt buffer (100mM Tris Cl pH 7.4, 10mM EDTA pH 8, 1M NaCl, 0.05% (v/v) Tween 20), and resuspended in High salt buffer. Labelled RNA was denatured by heating to 65 C for 5 minutes and placing on ice for 2 minutes. 10 μL High salt buffer was added to labelled RNA. Prewashed beads were placed on magnet, supernatant discarded, and then beads were resuspended in the labelled RNA. Beads and RNA were rotated for 30 minutes in the dark. Beads were then washed 4X for 1 minute with high salt buffer. Labelled RNA was eluted 2X with 100 mM DTT (1,4-Dithiothreitol). RNA was cleaned up with microelute columns (Norgen). Sequencing libraries were prepared from 250 ng of labelled RNA using the TruSeq Stranded Total RNA sequencing kit with RiboZero rRNA depletion, shortening the fragmentation step to 3 minutes and using 8 cycles of PCR amplification.