droplet-small-seq · High-throughput droplet-based single-cell small RNA sequencing technology
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2021-10-01 → 2023-11-29
- EU contribution
- €146,112
- Participants
- 1
- Scheme
- MSCA-IF
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Results in brief
High-throughput droplet-based single-cell small RNA sequencing technology
In biomedical and life sciences, single-cell RNA sequencing (scRNA-seq) has become widely popular for its ability to profile thousands of individual cells per experiment, revolutionizing the molecular identification and classification of cell types, cellular states, and rare phenotypes. However, most scRNA-seq methods rely on nascent poly-A tail capture, which restricts their utility to long RNAs such as protein coding and poly-A containing long non-coding RNAs. Though a few protocols have been proposed to sequence small RNAs (or total RNA) in single cells, most of them are either low-throughput or require highly sophisticated microfluidics, thus limiting their broader use in the field. Given the current technological state, numerous unanswered questions persist regarding the role of small RNA in developmental and pathological processes. For example, are small RNAs specific to a given cell type, and if so, what are their targets? How much is the expression of small RNAs variable between the same cell type? The potential clinical and therapeutic implications of finding answers to these questions are significant, as small RNAs can be targeted to manipulate cellular functions involved in disease pathogenesis. Given the biological importance of non-coding RNAs, the main objective of this project was to develop a high-throughput droplet-based scRNA-seq technology and apply it to investigate the regulatory roles of miRNAs. The small RNA sequencing technology established during the course of this project was successfully applied on leukemia cell model, demonstrating its proof-of-principle. While ongoing efforts are dedicated to its application in primary cells, we have already gained broad insights by employing single-cell long RNA-seq with inDrops-2, followed by trajectory analysis and in silico miRNA-target prediction. We identified potential miRNAs targeting hematopoietic lineage-specific genes and valuable insights in possible regulation of hemopoiesis.
Data: CORDIS, © European Union
Project objective
Droplet-based single-cell RNA-sequencing (scRNA-seq) technologies have penetrated almost all branches of life sciences and have significantly advanced our understanding of cellular processes and organism development. However, despite their astonishing impact, most of the scRNA-seq technologies reported to date rely on poly(A) tail capture and thus are mainly restricted to the protein-coding RNAs, while neglecting a substantial proportion of the transcriptome, including small non-coding RNAs. As a result, very little is known about the non-coding RNA expression and function in individual cells, and especially their role in the establishment of cellular phenotypic diversity. Small RNAs contain a variety of classes, of which miRNAs are the most common and these act as regulatory molecules by suppressing translation of mRNAs. In addition, loss-of-function studies of miRNAs uncovered their involvement in development of nearly all tissues, including hematopoiesis. However, most studies exploring miRNA dynamics reported to date relied on bulk cell assays, thus disregarding the individual cell types and their heterogeneity. In the scope of this proposal, we aim to develop a high-throughput droplet-based single-cell small RNA-seq (droplet-small-seq) for simultaneous miRNA and mRNA capture and sequencing. We will apply this newly developed technique to investigate the regulatory roles of miRNAs in cell fate decision during hematopoietic development at a single-cell level.
Original text from CORDIS.
Participants
- VILNIAUS UNIVERSITETAS · VilniusCoordinatorLithuania
Links
Data: CORDIS, © European Union