eSHAPE Total RNA Kit plus Data Analysis

Description

SHAPE RNA Structure Probing for Total RNA

An easy and fast protocol that allows the user to obtain in vitro or in cellulo SHAPE RNA Structure Probing data by NGS through NAI structure probing of Total RNA

eSHAPE total RNA is a method to obtain SHAPE RNA structure probing data for total RNA in vitro or in cellulo using the NAI reagent. eSHAPE total RNA is available as a kit for end user completion, and as a full service offering with results delivered as a data package containing RNA structure information as reactivity values.

• Compare total RNA from different sources to identify regions of RNA folding variability
• Reveal differences in RNA folding across different conditions or due to RNA binding factors
• Combine in vitro and in cellulo data to obtain delta SHAPE information for identifying positions interacting with RBPs
• Extremely deep RNA coverage >10,000X with minimal sequencing depth
• Elevated mutation rates in NAI samples compared to the DMSO control identify RNA positions with high reactivity/unpaired bases
• Reactivity values are predictive of paired and unpaired base status in RNA secondary structure folds

Eclipse Bio´s SHAPE RNA Structure Probing technology on Total RNA measures RNA flexibility at single nucleotide resolution for prediction of RNA secondary structures, in both cellular conditions (in cellulo) and purified, deproteinized RNA (in vitro). In vitro SHAPE reveals flexibility across RNA strands. In cellulo shows flexibility in regions not bound by proteins, which can help predict RNA’s secondary structure. Combining in cellulo and in vitro mutation rates, another method called ΔSHAPE (delta SHAPE), can identify which RNA positions interact with RNA binding proteins.

Selective 2’-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) is a chemical probing method that measures RNA flexibility at single nucleotide resolution. The licensed use of the patented reagent NAI (2-methylnicotinic acid imidazolide) in the RNA probing reaction forms adducts with the free 2′-OH on the RNA backbone in single stranded regions. During the reverse transcription reaction these adducts induce mutations in the newly formed cDNA at a higher rate than the DMSO control sample. Mutation rates are determined by NGS and calculated for each position of the RNAs. Computing into SHAPE reactivities guides the folding of the RNAs by indicating the pairing status for each position.

Sample Input: 1 ug Total RNA

Kit contents:

– RNA probing reagents incl. NAI for 8 samples
– DMSO for 8 control samples
– All enzymes, primers, adapters, and buffers needed to process 8 samples/to construct 16 NGS Libraries (8 NAI libraries and 8 DMSO libraries)
– Size selection beads
– Index primers

The Eclipse BioInnovations eSHAPE Total RNA Data Analysis Package delivers the results using Fastq files of the raw sequencing reads for each NAI library and DMSO library obtained by you as follows:

FASTA

BAM

SHAPE

QC Metrics

Coverage Plots

Mutation Rate Plots

Reactivity Plots

Reactivity Comparison Plots

Statistical Report