Abstract Number: PB1012
Meeting: ISTH 2021 Congress
Background: Zebrafish have two populations, young and mature thrombocytes. The mechanism of maturation of young to mature thrombocytes is not fully understood. We believe studying thrombocyte maturation might shed light on megakaryocyte maturation without the interference of polyploidy.
Aims: To perform single-cell RNA sequencing of young and mature thrombocytes in zebrafish.
Methods: Young (RFP+) and mature (GFP+) thrombocytes from GloFli fish were sorted separately in a 5 ml culture tube using BD FACSCanto flow cytometer. The instrument was set at 4°C with a nozzle size of 100. We set 75% to 90% cell viability with at least 10,000 cells for optimum analysis. The sorted cell samples were kept on ice and sent immediately to the Next Generation Sequencing core. RNA from these cells was prepared according to 10x Genomics protocols and sequencing was performed after successful library preparation and quality control.
Results: We used 2,176 RFP+ young thrombocytes and 1,541 GFP+ mature thrombocytes that survived. The total number of genes detected for GFP+ cells is 8,746 and for RFP+ cells, it is 6,990 genes. RNA-seq analysis of this data showed 6593 genes are expressed in both young and mature thrombocytes. Whereas mature thrombocyte uniquely expresses 2153 genes, mature thrombocyte expressed about 397 genes exclusively. About 80% of total genes in both GFP+ and RFP+ thrombocytes had human orthologous. The heatmap showed patterns that are consistent with the results mentioned above. We also analyzed the RNA-seq data by PANTHER program and found the gene-specific transcriptional regulators in the young and mature thrombocytes to be 14 and 140 genes, respectively. We also noted other differences.
Conclusions: RNA-seq analysis of young and mature thrombocytes identified differences in expression genes in various categories, including transcriptional regulators. This data should be useful in genome-wide knockdown screening to understand thrombocyte maturation.
To cite this abstract in AMA style:Fallatah W, Burks D, Azad R, Jagadeeswaran P. Single-cell Transcriptomics of Young and Mature Thrombocytes in Zebrafish [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/single-cell-transcriptomics-of-young-and-mature-thrombocytes-in-zebrafish/. Accessed November 30, 2021.
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