GSM5740012: SHAPE-MaP of NAI-treated NRAS-Luciferase, Replicate #2; Homo sapiens; OTHER
GSM5740012
Then all the transcripts were purified using Monarch RNA Cleanup Kit (NEB). The concentration was determined using a Nanodrop. Integrity and size of each transcript were confirmed using a 1% agarose gel. In vitro RNA folding and probing were carried out as previously described. Briefly, 2 ug of NRAS-G4-FL RNA in 89ul nuclease free H2O was heated at 95°C for 2 min, then immediately transferred to ice for 1 min. Then 10 ul of ice-cold RNA Folding Buffer 10X (500 mM HEPES pH 7.5; 1 M KCl) were then added, the solution mixed, and incubated at 37°C for 15 min. Then, 1 ul of 1 M MgCl2, pre-warmed at 37°C, was added, the solution mixed, and further incubated for 15 min. Probing was performed by adding 11 ul of NAI (1M stock), incubating at 37°C with moderate shaking for 10 min. For the control reaction, 11ul of neat DMSO was added. Reaction was quenched by adding 111ul of 1M DTT. RNA was recovered by purification on a Monarch RNA 10ug column (New England Biolabs). Both DMSO- and NAI-treated samples, were fragmented in a buffer containing 4mM final MgCl2, by incubating at 94°C for 8 min. The fragmented RNA was then subjected to random-primed reverse transcription, as per standard SHAPE-MaP conditions. Briefly, fragmented RNA was mixed with 1ul of 10uM random decamers, and 1ul 10mM dNTPs, then incubated at 70°C for 5 min, and immediately transferred to ice for 1 min. Then, 4 ul of 5X RT Buffer (250mM Tris pH 8.0; 375mM KCl), 2 ul of DTT (0.1M), 1 ul of SuperaseIN RNase inhibitor (Ambion), 1 ul of SuperScript II RT (ThermoFisher Scientific), and 1 ul 120mM MnCl2 were added, the reactions mixed, and incubated at 25°C for 10 min, followed by 2 hours at 42°C. After reaction cleanup, the buffer was replaced with the standard SuperScript II RT First Strand Buffer with MgCl2, and the RNA-cDNA hybrids were used as input for the NEBNext® Ultra™ II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs). After having converted the RNA into dsDNA, the remainder of the library preparation was then carried out using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs), as per manufacturer instructions.