GSM4202010: 2-12-minus-T25; Homo sapiens; OTHER
Cells were washed in ice cold PBS prior to incubation in Permeabilization Buffer (1mL/10^6 cells; 100μL/10^7 cells; 10mM Tris-HCl pH 7.4, 200mM sucrose, 10mM KCl, 5mM MgCl2, 1mM EDTA, 0.05% v/v Tween20, 0.15% v/v NP-40, 0.5mM DTT, 2U/mL SUPERase•In (ThermoFisher Scientific, AM2694) containing Roche cOmpleteTM protease inhibitors for 5 minutes on ice. The cell pellet was washed twice with Permeabilization Buffer (0.5mL/10^6 cells per wash) and then resuspended in Storage Buffer (10mM Tris-HCl pH 8, 25% glycerol, 5mM MgCl2, 0.1mM EDTA, 5mM DTT). For the nuclear run-on reaction, permeabilized cells were incubated with one volume of 2X Nuclear Run-On reaction mastermix containing Biotin 11-CTP (1.4mM Tris-HCl pH 8, 0.7mM MgCl2, 0.14mM DTT, 42mM KCl, 0.05mM Biotin 11-CTP (Perkin Elmer NEL544001EA), 2.5µM CTP, 0.25mM ATP, 0.25mM GTP, 0.25mM UTP, 40U SUPERase•In, 1% Sarkozyl (Sigma Alrich L5125)) at 37ºC for three minutes with gentle agitation prior to the addition of TRIzol-LS (ThermoFisher 10296010). TRIzol samples were mixed with chloroform and the aqueous phase was isolated after centrifugation. 1μL GlycoBlue and 2.5 volumes ethanol was added to the aqueous phase for RNA precipitation (incubation at room temperature for 10 minutes, 14000g centrifugation at 4ºC for 20 minutes). Precipitated RNA was washed with 70 percent ethanol, resuspended in nuclease free water. RNA was denatured at 65ºC for 40 seconds and then was incubated on ice with 0.2M NaOH for 12.5 minutes prior to the addition of 1 volume 1M Tris-HCl (pH 6.8). Base hydrolyzed RNA was exchanged to 10mM Tris solution using a Micro Bio-Spin 30 Column (BIORAD, 7326250) as per the manufacturer's instruction. For enrichment of Biotin-incorporated RNA, hydrolyzed RNA was incubated with Dynabeads M-280 Streptavidin (ThermoFisher Scientific, 11206D) in PRO-Seq Binding Buffer (10mM Tris-HCl pH 7.4, 300mM NaCl, 0.1% v/v TritonX-100) at room temperature for 20 minutes with gentle rotation. Streptavidin beads were washed (1 minute with rotation per wash) three times each in High Salt Wash Buffer (50mM Tris-HCl pH 7.4, 2M NaCl, 0.5% v/v TritonX-100), Binding Buffer and Low Salt Wash Buffer (5mM Tris-HCl pH 7.4, 0.1% TritonX-100) and then resuspended in TRIzol. Following incubation at room temperature for 3 minutes, TRIzol samples were mixed with chloroform and the aqueous phase was isolated after centrifugation. TRIzol/chloroform treatment of washed Streptavidin beads was repeated once and beads were incubated with chloroform for 3 minutes at room temperature prior to centrifugation. 1μL GlycoBlue and 900μL ethanol was added to the pooled aqueous phase solution and samples were incubated at room temperature for 10 minutes prior to centrifugation. Precipitated biotin-enriched RNA was washed with 70% ethanol and resuspended in nuclease free water. PRO-Seq Sequencing libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit with Ribo-Depletion NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina
GEO Accession
GSM4202010