GSM8014374
10 million cells were lysed with 4 mL ice-cold hypotonic buffer (10 mM Tris-Cl pH 7.5, 10 mM NaCl, 2.5 mM MgCl2, 0.5% NP-40) on ice for 5 min. The cell lysate was carefully underlaid with 1 mL sucrose cushion buffer (hypotonic buffer with 10% (w/v) sucrose). The nuclear fraction was pelleted by centrifugation (300 g, 5 min at 4 oC), then washed with ice-cold hypotonic buffer (without NP-40) once. The purified nuclear fraction was first resuspended in 300 µL (200~500 µL, depending on the pellet size) ice-chilled NUN1 buffer (20 mM Tris-HCl pH 7.9, 75 mM NaCl, 0.5 mM EDTA pH 8.0, and 50% glycerol) and then lysed in 3 mL (at least 10-fold volume of NUN1) ice-cold NUN2 buffer (20 mM HEPES-KOH pH 7.6, 300 mM NaCl, 0.2 mM EDTA pH 8.0, 7.5 mM MgCl2, 1% NP-40, 1 M Urea, 3% Empigen BB, 1x Protease inhibitor Complete EDTA-free, and 1x PhosSTOP). Note: The cotton candy-like chromatin should appear within a few minutes. The chromatin was then pelleted and washed with ice-cold PBS once (300 g, 1 min at 4 oC). Chromatin-bound Pol II was released by DNase digestion in the reaction mixture (10 mM Tris-HCl pH 7.5, 400 mM NaCl, 100 mM MnCl2, 2 Units/ µL RiboLock, and 0.2 Units/µL Turbo DNase) at 37oC for 15 min in the ThemoMixer C (Eppendorf) at 1200rpm. EDTA pH 8.0 (final 1mM) was used to stop the DNase reaction, and the supernatant was collected by high-speed centrifugation (14,000g, 10 min at 4oC). A 5-fold volume of ice-cold NET-2E buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05 % NP-40, and 3% Empigen BB) was used for the dilution of the supernatant.  The 10 µg of anti-Pol II antibody MABI601 were mixed with 200 µL anti-mouse IgG Dynabeads solution in 1 mL NET-2 buffer (NET-2E without Empigen) and incubated at 4 oC with mild rotation for at least 1 hour. The conjugated beads were washed with NET-2 buffer once and resuspended in 200 µL NET-2E buffer. The resuspended beads were added to the diluted supernatant of chromatin for at least 1 hours and then incubated at 4oC with mild rotation (Note: Avoid overnight incubation). After the incubation, beads were washed with 1 mL of ice-cold NET-2E buffer at least 6 times. Pol II-bound nascent RNAs (POINTs) were extracted from the beads using Tri Reagent and subsequently purified by Direct-zol kit following the manufacturer's protocol.  Equal amount of SIRV spike-in oligos were added to each POINTs. NEBNext Ultra II Directional RNA library prep kit