title: GSM8217170: MYCOE_2; Homo sapiens; OTHER
library_construction_protocol: For nuclei isolation, cells were incubated with swelling buffer (10 mM Tris-Cl pH 7.5, 2 mM MgCl2 and 3 mM CaCl2) for 5 min on ice and then incubated with lysis buffer (swelling buffer with 0.5% NP-40 and 10% glycerol) for 5 min on ice, before being re-suspended in 100 μl of freezing buffer (50 mM Tris-Cl pH 8.0, 40% glycerol, 5 mM MgCl2 and 0.1 mM EDTA). For the run-on assay, an equal volume of reaction buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 300 mM KCl, 1 mM dithiothreitol, 20 units of SUPERase•In, 1% sarkosyl, and 500 μM ATP, GTP, bio-UTP and bio-CTP) was added into each sample before incubation at 30 °C for 5 min. The nuclear run-on RNA was then extracted with TRIzol LS reagent (10296010, Invitrogen) and subjected to hydrolysis, buffer exchange and purification by streptavidin beads (88816, Thermo Fisher). Purified RNA was treated with PNK before being used for complementary DNA synthesis by using the NEBNext Multiplex Small RNA Library Prep Set for Illumina Kit (E7300S, NEB). Obtained complementary DNA template was amplified by PCR using the Phusion High-Fidelity enzyme (M0530L, NEB) for deep sequencing.