GSM8338095: PGK, PRO-seq, rep1; Homo sapiens; OTHER
To prepare nuclei, cells were scraped from 70% confluent 10 cm plates in ice-cold PBS. A portion (20%) was taken for RNA-seq library preparation. Cells were then pelleted (700g, 4°C, 3 min), resuspended in permeabilization buffer (10 mM Tris Cl pH 7.5, 10 mM KCl, 250 mM sucrose, 5 mM MgCl2, 1mM EGTA, 0.05% Tween 20, 0.5 mM DTT), incubated on ice for 10 min, pelleted, washed (10 mM Tris Cl pH 7.5, 10 mM KCl, 150 mM sucrose, 5 mM MgCl2, 0.5 mM CaCl2, 0.008% Tween 20, 0.5 mM DTT), resuspended in storage buffer (50 mM Tris Cl pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT), snap frozen and stored at -80°C. The nascent RNA was generated using biotin run-on reactions, extracted using the Norgen Total RNA Purification Kit, and hydrolyzed using NaOH. A 3′ adapter (rUrNrNrNrNrNGATCGTCGGACTGTAGAACTCTGAAC/3InvdT/ ) was ligated to the nascent RNA, purified with streptavidin beads, 5′ ends were modified, and a 5′ adapter (/5InvddT/CCTTGGCACCCGAGAATTCCANrNrNrNrNrNrC) ligated. RNA was purified with Trizol, reverse transcribed, PCR amplified, and PAGE-purified.