Plasmids developed in the lab are available through Addgene.
DART-seq
The DART-seq method enables single-nucleotide mapping of m6A sites transcriptome-wide from low amounts of input RNA. DART-seq works by using a fusion protein consisting of the m6A-binding YTH domain tethered to the C-to-U deaminase APOBEC1. This APOBEC1-YTH fusion protein binds to m6A sites and targets nearby cytidine residues for C-to-U deamination. Since nearly all m6A sites are followed by a cytidine, DART-seq can identify m6A residues with single-nucleotide resolution.
DART-seq offers several advantages, including its compatibility with virtually all RNA-seq platforms and its ability to profile m6A transcriptome-wide from ultra-low amounts of input RNA. Additionally, DART-seq can be performed either by expressing APOBEC1-YTH in cells or by mixing an RNA sample of interest with purified APOBEC1-YTH protein in an in vitro DART reaction.
Publications and Protocols:
Meyer KD. DART-seq: an antibody-free method for global m6A detection. Nature Methods. 2019. Link to paper.
Tegowski M, Zhu H, Meyer KD. Detecting m6A with In Vitro DART-Seq. Methods in Molecular Biology book series. 2022. Download the protocol.
single-cell DART-seq (scDART-seq)
The DART-seq method can be applied to single cells to achieve single-cell m6A profiling using scDART-seq. While this method is currently optimized using cells that express the APOBEC1-YTH protein, we are hoping to further develop in vitro DART-seq for single-cell m6A mapping in the future.
Publications and Protocols:
Tegowski M, Prater AK, Holley CL, Meyer KD. Single-cell m6A profiling in the mouse brain uncovers cell type-specific RNA methylomes and age-dependent differential methylation. Nature Neuroscience. 2024 Sep 24. Read it here.
Tegowski, M, Flamand, MN, Meyer, KD. scDART-seq reveals distinct m6A signatures and mRNA methylation heterogeneity in single cells. Molecular Cell. 82 (4): 868-878, 2022. Read it here.
Tegowski, M and Meyer, KD. Detection of m6A in single cultured cells using scDART-seq. STAR Protocols. 3 (3) 16 Sep 2022. Link to protocol.
TRIBE-STAMP
This method enables simultaneous identification of the RNA targets of two RBPs in cells. To do this, two RBPs of interest are fused to the ADAR or APOBEC1 enzymes to enable A-to-I and C-to-U editing, respectively, of target RNAs.
Publications:
Flamand, MN, Ke, K, Tamming, R, and Meyer, KD. Single-molecule identification of the target RNAs of different RNA binding proteins simultaneously in cells. Genes and Development 2022. Read it here.
Flamand M, Meyer KD. Simultaneous profiling of the RNA targets of two RNA-binding proteins using TRIBE-STAMP. Methods in Enzymology. 2024. In press. Humana, New York, NY. Read it here.
Bullseye Pipeline
Analysis of DART-seq, scDART-seq, and TRIBE-STAMP datasets can be done using Bullseye, a pipeline we developed for identification of C-to-U and A-to-I editing events. The publications listed above for each of these methods describe details for Bullseye settings used for that method. Additional resources for using Bullseye are available at the Bullseye GitHub page.
Access the Bullseye GitHub page here.
DART-FISH
DART-FISH leverages the m6A-dependent C-to-U editing of DART-seq to enable in situ visualization of individual m6A sites in cellular RNAs of interest. By coupling DART protein expression with mutation-sensitive FISH, this method allows simultaneous visualization of the methylated and unmethylated copies of specific transcripts in cells.
Protocols:
Sheehan CJ, Marayati BF, Bhatia J, and Meyer KD. In situ visualization of m6A sites in cellular mRNAs. Nucleic Acids Research. 2023. Read it here.
Sheehan CJ, Meyer KD. Simultaneous in situ detection of m6A-modified and unmodified RNAs using DART-FISH. Methods in Molecular Biology. 2024. Read it here.
Genetically Encoded m6A Sensor System (GEMS)
GEMS is the first method for sensing m6A in living cells. The system couples mRNA methylation with EGFP fluorescence, enabling a simple readout for m6A which is compatible with high throughput screening applications. Furthermore, GEMS is entirely genetically encoded, which facilitates its use in a wide variety of tissue or cell types and even in vivo. In addition to its utility as a readout for mRNA methylation, GEMS also serves as a versatile platform for expressing proteins of interest in an m6A-dependent manner.
Publications:
Marayati BF, Thompson MG, Holley CL, Horner SM, Meyer KD. Programmable protein expression using a genetically encoded m6A sensor. Nature Biotechnology. 2023. Read it here.