APOBEC1-YTH-HA and APOBEC1-YTHmut-HA proteins were in vitro transcribed/translated using the Promega TNT T7 Quick Coupled In Vitro Transcription/Translation kit. Briefly, 1 μg of plasmid DNA was used in a 50 μl reaction and incubated for one hour at 30°C. 5 μl of each reaction was then mixed with 30 ng of a 1500 nt-long RNA with a single internal A or m6A site, 0.5 μl RNasin (Promega), in 1X deaminase buffer (10mM Tris-HCl pH7.5, 50mM KCl, 0.1uM ZnCl2). Reactions were incubated for 4 h at 37°C. RNA was isolated with the Qiagen RNeasy Plus Mini kit and treated with DNase I (New England Biolabs) for 15 min at 37°C. For assays using cellular RNA, in vitro deamination was carried out for 6 h at 37°C using 50 ng of total RNA from HEK293T cells. Sequencing libraries were then prepared using the NebNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).
Rneasy plus mini kit
Manufactured by New England Biolabs
The RNeasy Plus Mini kit is a tool for the purification of total RNA from various sample types. It utilizes a silica-membrane-based technology to efficiently capture and purify RNA molecules, allowing for the isolation of high-quality RNA for downstream applications.
Automatically generated - may contain errors
2 protocols using rneasy plus mini kit
1
In vitro Deamination Assay for APOBEC1
2
In vitro Deamination Assay for APOBEC1
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APOBEC1-YTH-HA and APOBEC1-YTHmut-HA proteins were in vitro transcribed/translated using the Promega TNT T7 Quick Coupled In Vitro Transcription/Translation kit. Briefly, 1 μg of plasmid DNA was used in a 50 μl reaction and incubated for one hour at 30°C. 5 μl of each reaction was then mixed with 30 ng of a 1500 nt-long RNA with a single internal A or m6A site, 0.5 μl RNasin (Promega), in 1X deaminase buffer (10mM Tris-HCl pH7.5, 50mM KCl, 0.1uM ZnCl2). Reactions were incubated for 4 h at 37°C. RNA was isolated with the Qiagen RNeasy Plus Mini kit and treated with DNase I (New England Biolabs) for 15 min at 37°C. For assays using cellular RNA, in vitro deamination was carried out for 6 h at 37°C using 50 ng of total RNA from HEK293T cells. Sequencing libraries were then prepared using the NebNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs).
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