SHAPE-MaP experiments were performed with the same cell lines (above) on natively transcribed mRNAs. RNA was extracted by standard TRIzol purification and DNase digestion (ThermoFisher TRIzol, 5PRIME PhaseLock Heavy, Invitrogen Purelink RNA columns, ThermoFisher Purelink DNase Set) and incubated at 37°C for 10 min in folding buffer (above, in vitro SHAPE treatment). SHAPE treatment was performed for 5 min with an addition of DMSO or three separate additions of 1M7 with a final concentration of 30 mM 1M7, 10% DMSO. We performed buffer exchange (GE illustra MicroSpin G-50 Columns) before depletino of ribosomal RNA (RiboMinus Eukaryote System v2 from Life Technologies).
5prime phaselock heavy
Manufactured by Thermo Fisher Scientific
The 5PRIME PhaseLock Heavy is a laboratory equipment product. It is designed to facilitate the separation and isolation of nucleic acids, proteins, and other biomolecules during various experimental procedures.
Automatically generated - may contain errors
2 protocols using 5prime phaselock heavy
1
SHAPE-MaP Analysis of Natively Transcribed mRNA
2
Bicine Buffer Treatment and RNA Extraction
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Cells were grown in 10-cm plates and grown to 80%–90% confluency. Cells were washed with PBS and replenished with 1350 µL of media supplemented with 200 mM bicine (pH 8.0) buffer (final concentration). A total of 150 µL 5-nitroisatoic anhydride (5NIA) at 250 mM in dimethyl sulfoxide (DMSO) was added. Controls were treated with 150 µL DMSO. Cells were then incubated at 37°C for 10 min. RNA was extracted by TRIzol purification and DNase digestion (Thermo Fisher TRIzol, 5PRIME Phase Lock Heavy, Invitrogen Purelink RNA columns, Thermo Fisher Purelink DNase Set) and quantified with a NanoDrop spectrophotometer.
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