Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 1562, 231-243

High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq)

Affiliations

High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq)

Eva Hrabeta-Robinson et al. Methods Mol Biol.

Abstract

N 1-methyladenosine (m1A), N 3-methylcytidine (m3C), and N 1-methylguanosine (m1G) are common in transfer RNA (tRNA) and tRNA-derived fragments. These modifications alter Watson-Crick base-pairing, and cause pauses or stops during reverse transcription required for most high-throughput RNA sequencing protocols, resulting in inefficient detection of methyl-modified RNAs. Here, we describe a procedure to demethylate RNAs containing m1A, m3C, or m1G using the Escherichia coli dealkylating enzyme AlkB, along with instructions for subsequent processing with widely used protocols for small RNA sequencing.

Keywords: AlkB; N 1-methyladenosine (m1A); N 1-methylguanosine (m1G); N 3-methylcytidine (m3C); RNA Sequencing; RNA demethylation; Transfer RNA (tRNA).

Figures

Figure 1
Figure 1
Typical SDS-PAGE analysis of purified AlkB protein.
Figure 2
Figure 2
Typical BioAnalyzer Trace of human small RNA preparation after DNase and AlkB treatment, 1:20 dilution.
Figure 3
Figure 3
Typical size selection of cDNA libraries adapted for Illumina sequencing using 2% Agarose Size Select E-gel. Photographs are taken 10 minutes after starting the run (a), and after the collection of the desired fraction (b). A 50 bp E-gel ladder was used as a guide for fraction collection.
Figure 4
Figure 4
Typical BioAnalyzer trace of 1:10 dilution of cDNA library used as input for Illumina sequencing in ARM-seq.

Similar articles

See all similar articles

Cited by 3 articles

Publication types

LinkOut - more resources

Feedback