RNA circularization protocol

RNA circularization

"GTP was added to a final concentration of 2 mM along with a buffer that included magnesium (50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT; Thermo Fisher)."
——bioRxiv, 2022

"Optionally, after DNase I digestion, GTP was added to the reaction at a final concentration of 2 mM, and then the reactions were incubated at 55°C for 15 min to catalyze the cyclization of circRNAs."
——Cell, 2022

For circRNA, after DNase treatment additional GTP was added to a final concentration of 2 mM, and then reactions were heated at 55 °C for 15 min. RNA was then column purified.
In some cases, purified RNA was recircularized: RNA was heated to 70 °C for 5 min and then immediately placed on ice for 3 min, after which GTP was added to a final concentration of 2mM along with a buffer including magnesium (50mM Tris-HCl, 10mM MgCl2, 1mM DTT, pH 7.5; New England Biolabs). RNA was then heated to 55 °C for 8 min, and then column purified.
——Nature Comm, 2022

50 ug circRNA precursors were heated to 70°C for 3 min and immediately placed on ice for 2 min, then GTP was added to a final concentration of 2 mM with a buffer that has the same components as the T4 RNA ligase buffer (NEB) for 8 min at 55°C .
——Molecular cell, 2022

Transcription templates were removed with TurboDNase as per manufacturer’s instructions, and post-transcriptional circularization was facilitated by supplementing each reaction with 2 mM GTP and incubating at 55◦C for 15 min, followed by rapid cooling to 4◦C. RNA was purified using a MEGAclear kit (circPVT1;Ambion) or by ammonium acetate/ethanol precipitation (cSAA) according to MEGAshortscript instructions.
——Nucleic Acids Research, 2021

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NEB: T4 RNA Ligase 1 (ssRNA Ligase)
Catalog #: M0204S , M0204L

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可单独购买：T4 RNA Ligase Reaction Buffer Catalog #: B0216L

其他常见商业化缓冲液

NEBuffer™ 2
Catalog #: B7002S

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NEBuffer™ 1.1

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NEBuffer™ 2.1

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NEBuffer™ 3.1

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rCutSmart™ Buffer

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ABclonal EcoRI

1X Buffer CutS 组成
50 mM KAc, 20 mM Tris-HAc, 10 mM MgAc2, 100 μg/mL
BSA, pH 7.9 @ 25°C

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内切酶储存缓液：

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稀释液 B

ssRNA Ladder
Catalog #: N0362S