Most successful epitranscriptomics studies rely on large amounts of input sample; however, huge findings may also reside in the smallest of specimens, such as zygotes or embryos. Now, a study provides exciting in vivo insight into these microcosms by describing the development of an epitranscriptomic mapping technique for single-cells and rare cell types.
While traditional N6-methyladenosine (m6A) mapping techniques such as MeRIP–seq and m6A-seq require large amounts of RNA, techniques such as DART-seq/single-cell DART-seq need relatively small amounts of input samples; however, these advanced techniques only apply to cultured cells. Now, a “small” team of researchers led by Kin Fai Au, Arne Klungland, and John Arne Dahl describe “picogram-scale m6A RNA immunoprecipitation and sequencing” (picoMeRIP-seq) as an alternative m6A mapping technique that can be applied to single zygotes, oocytes, and preimplantation embryos.
Let’s hear more on this very tiny-yet-titanic advance in m6A mapping technology from Li and colleagues:
- Optimization of picoMeRIP-seq involves enhancing sample recovery and signal-to-noise ratio, identifying an optimal anti-m6A antibody, developing an RNA fragmentation procedure (sonication), and tailoring a straightforward/rapid library preparation protocol (Takara Bio’s SMART technology) to a single-tube procedure
- picoMeRIP-seq employs standard laboratory equipment and reveals consistent profiles between replicates and different starting sample amounts (10 nanograms, 1 nanogram, and 100 picograms of mouse liver RNA)
- picoMeRIP-seq in zebrafish zygotes demonstrates similar m6A profiles for single and pooled zygotes and replicates of single zygotes, with detected m6A peaks from single zygotes close to the expected maximum
- picoMeRIP-seq of mouse embryonic stem cells reveals similar data from 1000 and 10 cells, with high reproducibility (although single cells did not allow the generation of sufficient sequencing libraries)
- Turning to mouse development, single-oocyte and single-embryo m6A data contain sufficient information for clustering according to cell identity and distinguishing closely related oocyte and embryo stages
- Genes marked by m6A in certain oocyte and embryo stages display enrichment in gene ontology terms such as cell proliferation, apoptosis, RNA splicing, and embryonic development
This small-yet-substantial study reports how picoMeRIP-seq can generate epitransciptomic maps from tiny amounts of input RNA without requiring specialized equipment. The authors foresee that this epitransciptomic mapping technique will help researchers across the globe to describe the epitransciptomic roots of disease (by analyzing healthy and diseased tissue biopsies) and fertility and developmental defects (by analyzing human oocytes and preimplantation embryos).
For more on the minuscule-yet-massive epitranscriptomic mapping potential of picoMeRIP-seq, see Nature Biotechnology, June 2023.