Development of single-cell lineage tracing technique based on random barcode in zebrafish embryos

Abstract

Sequencing of transcribed clonally encoded random barcodes(TracerSeq) is a single cell RNA sequencing based method to trace lineage of cells from early developmental stage. By Tol2 integration of injected random barcode into genome of zebrafish, cell lineage can be traced using by each integration events. Previous study using TracerSeq was conducted with few embryos of same developmental time point, with no discrimination of individuals. In this study, TracerSeq random barcode library was evolved on its structure by library ID and on cloning efficiency. I confirmed the increased efficiency of barcode integration by performing bulk cDNA Oxford Nanopore sequencing on embryos injected with barcodes. A novel idea of utilizing TracerSeq on a transgenic line and performing cell lineage tracing using barcodes in FACS-sorted cells was proposed, specifically targeting a particular organ or lineage to gain more detailed understanding of the cell fate decision process. Random barcode injected transgenic Sox10:Gal4;UAS:EGFP zebrafish embryos which express strong green fluorescence in early neural crest cells. Among random barcoded embryos, 8 embryos of various developmental stages were collected and FACS sorted. Double positive cells which means neural crest cells that has barcode integrated in its genome was collected. While the cell population was not sufficient to perform single-cell RNA sequencing, qPCR analysis of these cells revealed the expression of neural crest markers. Therefore, it is foreseen that attaining a larger cell population will enable the acquisition of robust single-cell lineage tracing data.