Computational prediction of RNA structures using DMS-seq data
1. Raw DMS-seq data from different biological replicates were pooled after checking the reproducibility of the data (if applicable).
2. For the RNA region of interest, normalize the raw DMS-seq data to the most highly reactive residue within the region after removing outliers by 95% Winsorisation. In this procedure, all data above the 95th percentile is set to the 95th percentile.
3. A/C bases with a DMS-seq signal greater that 20% of the signal on the most highly reactive residue (after the normalization described in 2) were called ‘unpaired’. The signal cutoff can be further optimized by using the receiver operating characteristic (ROC) curve that compares the DMS-seq signal of the RNA to the structure model obtained by other approaches (e.g. published crystal structure, if available).
4. Generate the RNA sequence of the region of interest.
5. Generate the folding constraint, by assigning the experimentally determined unpaired A/C bases as “x” and the bases with no constraint as “.”.
7. The mRNA structural model was visualized using VARNA (http://varna.lri.fr/), by importing the RNA sequence and the structure prediction in dot-bracket notation where the characters “(” and “)” correspond to the 5’ base and the 3’ base in the base-pair, respectively, while “.” denotes an unpaired base.
Copyright: Content may be subjected to copyright.
How to cite:
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
Zhang, Y(2020). Computational prediction of RNA structures. Bio-protocol Preprint. bio-protocol.org/prep641.
Burkhardt, D. H., Rouskin, S., Zhang, Y., Li, G., Weissman, J. S. and Gross, C. A.(2017). Operon mRNAs are organized into ORF-centric structures that predict translation efficiency. eLife. DOI: 10.7554/eLife.22037
Post your question to gather feedback from the community. We will also invite the authors of this
article to respond.
0/150
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Spinning
Post a Question
0 Q&A
Spinning
This protocol preprint was submitted via the "Request
a Protocol" track.