title: GSM4478427: 293T_polysome_mono; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Cells were collected and lysed with lysis buffer (10 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 100 mM KCl, 1% Triton X-100, 2mM DTT, 100μg/ml CHX (CST), 50U/ml RNasin Plus RNase Inhibitor (Thermo fisher) and cOmplete EDTA-free protease inhibitor (1 mini tablet per 10ml, Roche)). 10–50% sucrose gradients were prepared in gradient buffer (20mM HEPES-KOH, pH7.4, 5mM MgCl2, 100mM KCl, 2mM DTT, 100μg/ml CHX, 50U/ml RNasin Plus RNase Inhibitor) using a Gradient Master (Biocomp). 400μl lysates were loaded on the gradients. Gradients were centrifuged at 36,000 rpm for 2 hours at 4 ℃ in an SW-40 Ti rotor and then fractionated using the Gradient Master. RNA was then isolated using TRIzol reagent. Polyadenylated RNA of each fraction was separated from total RNA using Oligo dT Magnetic Beads (Vazyme). 10-50 ng of polyadenylated RNA was converted using the EZ RNA methylation kit (Zymo Research) with 3 cycles of [70 C 10 min, 64 C 45 min] and 37 C 30 min for desulfonation. The converted RNA was fragmented into 150~200 nt fragments by incubation at 94 °C for 8 minutes in fragmentation buffer (NEBNext Ultra II Directional RNA Library Prep Kit, NEB). The fragmented RNA was then used for library construction, following the manufacturer's protocol (NEBNext Ultra II Directional RNA Library Prep Kit, NEB).
attributes.tag: GEO Accession
attributes.value: GSM4478427