Sep 25, 2022
dsRNAs treatment with RNase T1 and DNase I
- 11Département de Biologie, Centre SÈVE, Université de Sherbrooke, Sherbrooke, QC J1K 2R1, Canada
Protocol Citation: Vahid Jalali Javaran 2022. dsRNAs treatment with RNase T1 and DNase I. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqj9kyvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 16, 2022
Last Modified: September 25, 2022
Protocol Integer ID: 70142
Abstract
For dsRNA sequencing by nanopore sequencing, this protocol was used. Before treating samples with RNase T1, you should measure the total concentration of RNAs in the samples by using a nanodrop or Qubit device, as RNase T1 has the ability to partially digest double-stranded RNAs in the absence of single-stranded RNA.
Materials
00:00:00
1. DNase I (RNase-free)
2. DNase I Reaction Buffer (10X)
3. RNase T1
Digestion
Digestion
30m
30m
Add 10X DNase Buffer with MgCl2 (final concentration should be 1X).
Add 50 units RNase T1 per 1µg of total RNA and 1 unit DNase I per 2µg of total RNA
Incubate at 37 degrees C for 20 min.
Inactivation of enzymes
Inactivation of enzymes
cleanup with phenol/chloroform extraction.