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.
