Periodic Reporting for period 1 - droplet-small-seq (High-throughput droplet-based single-cell small RNA sequencing technology)
Berichtszeitraum: 2021-10-01 bis 2023-09-30
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.
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.
To achieve the objectives, we advanced single-cell RNA-seq (inDrops-2) platform, which was used as a basis for single-cell small RNA sequencing technology. In particular, we implemented updated microfluidics platform to achieve higher throughput and high hydrogel bead loading within stable droplet formation. We adjusted droplet volumes and hydrogel bead sizes to further enhance the performance. Following extensive testing of various small RNA capture strategies and rounds of optimization in both bulk and droplet formats, we implemented a method for single cell small RNA-seq. This method utilizes several consecutive enzymatic reactions to capture, barcode and amplify cDNA molecules, which can be size selected and consequently sequenced (Fig. 1a). For initial validation, we applied our method to quantify small RNAs on bulk and droplet formats of the leukemia K562 cell line. Using our approach, we were able to detect the expression levels of small RNAs, including miRNAs, tRNAs, snoRNAs and snRNAs, which map to their precursor gene sequences with a high precision (Fig. 1b). Furthermore, we simultaneously captured fragments of long RNAs, including protein coding and long non-coding RNAs, which provide additional information on gene expression in each cell. We detected ~3.5% of total reads mapping to precursor miRNA sequences (Fig. 1c). Overall, using our approach we captured approximately 80 to 370 unique small RNA molecules per cell (Fig. 1d), averaging to around 127 UMIs per cell (Fig. 1e). Additionally, we implemented and fine-tuned the bioinformatics analysis pipeline to process sequencing data obtained by our method. In parallel, we enhanced long RNA capture using inDrops-2 and assessed its performance with commercial 10X Genomics v3. Analysis of live and fixed peripheral mononuclear cells (PBMC) demonstrated comparable UMI and gene capture rates between the methods and cell preservation types, indicating the effectiveness of the inDrops-2 method. Further, we profiled single cell long RNAs of bone marrow progenitor (CD34+) cells using inDrops-2. Subsequently, we performed trajectory analysis to pinpoint lineage-specific genes and employed in silico miRNA-target prediction to further characterize these genes. The analysis revealed multiple miRNAs as potential regulators of hemopoiesis with conserved target sequences within the lineage-specific genes.
To reach our targeted audience, comprising scientists in the field, we presented the droplet-small-seq results at the “EMBO | EMBL Symposium: The non-coding genome” (Heidelberg, Germany). Results of the project will be shared via reputable, open-access, peer-reviewed journals. Presently, we have deposited a preprint to the bioRxiv for a manuscript detailing an improved scRNA-seq method (inDrops-2), which we also use as a basis for small RNA-seq. To gain experience in analyzing scRNA-seq data, the fellow contributed to a study deciphering microenvironment of clear cell renal cell carcinoma, which is also deposited on the bioRxiv. Although outside the scope of the project, the acknowledgment of the MSCA fellowship appears in a letter, which was written in response to authors who misinterpreted the fellow and his former colleagues' previous research and misapplied Mendelian Randomization analysis. Besides the previously mentioned manuscripts, we collected sufficient results that will be reported in at least one more publication. This publication will provide a detailed description of the single-cell small RNA-seq method and currently ongoing efforts for its application in primary cells such as peripheral PBMC and CD34+ cells.
Our project has attracted attention from international media outlets, such as GenomeWeb. To share his experiences about writing a successful MSCA application and to encourage others to apply for the fellowship, the fellow gave a talk at the “Baltic MSCA Postdoctoral Fellowships Inspiration Day” event, jointly organized by the Estonian, Latvian and Lithuanian Research Councils. Moreover, the project was also selected as one of the Widening Countries Inspirational Stories by the MSCA-NET.
To reach our targeted audience, comprising scientists in the field, we presented the droplet-small-seq results at the “EMBO | EMBL Symposium: The non-coding genome” (Heidelberg, Germany). Results of the project will be shared via reputable, open-access, peer-reviewed journals. Presently, we have deposited a preprint to the bioRxiv for a manuscript detailing an improved scRNA-seq method (inDrops-2), which we also use as a basis for small RNA-seq. To gain experience in analyzing scRNA-seq data, the fellow contributed to a study deciphering microenvironment of clear cell renal cell carcinoma, which is also deposited on the bioRxiv. Although outside the scope of the project, the acknowledgment of the MSCA fellowship appears in a letter, which was written in response to authors who misinterpreted the fellow and his former colleagues' previous research and misapplied Mendelian Randomization analysis. Besides the previously mentioned manuscripts, we collected sufficient results that will be reported in at least one more publication. This publication will provide a detailed description of the single-cell small RNA-seq method and currently ongoing efforts for its application in primary cells such as peripheral PBMC and CD34+ cells.
Our project has attracted attention from international media outlets, such as GenomeWeb. To share his experiences about writing a successful MSCA application and to encourage others to apply for the fellowship, the fellow gave a talk at the “Baltic MSCA Postdoctoral Fellowships Inspiration Day” event, jointly organized by the Estonian, Latvian and Lithuanian Research Councils. Moreover, the project was also selected as one of the Widening Countries Inspirational Stories by the MSCA-NET.
We anticipate that our contributions in the development of the state of the art single-cell sequencing technologies will substantially broaden the scope of single-cell transcriptomics, by offering a possibility of integrating the small RNA dimension with the existing protein-coding transcriptome. In particular, when it comes to small non-coding RNAs, many aspects of their biology in single cells are still unknown. For example, are small RNAs acting as drivers of cell differentiation or rather homeostasis regulators? What is the specificity of a given small RNA to a cell type and its impact on cellular phenotype? These questions, and many others, can be tackled using our method, and finding answers may have significant clinical and therapeutic implications in the long term, as small RNAs can be targeted to manipulate cell functions. Moreover, this technology can facilitate the identification of miRNA-target interactions specific to individual cells, inferred from expression dynamics in single-cell sequencing, allowing to measure how strong or weak the effect of miRNA regulation on a target might be. Furthermore, our platform could be adapted (with minor modifications) to other biological systems, such as microorganisms, whose RNA molecules lack polyadenylation, and thus could benefit broader areas of biotechnology, microbiology, and others.