ACME and SPLiT-seq: Quantitative and evolutionary single-cell transcriptomics in planarians

The last decade has seen an exponential development of scRNA-seq technologies. This thesis begins by recapitulating the history of transcriptomics, from its early years to the development of modern single-cell protocols, in order to understand the evolution and current state of the field. During the experimental part, I focus on the description and validation of a versatile pipeline for scRNA-seq. This combines a novel sample preparation strategy (ACME) with a powerful in situ barcoding platform (SPLiT-seq) to overcome some of the restrains of current single-cell protocols. ACME provides simultaneous tissue dissociation, fixation and permeabilization, resulting in cells that can be cryopreserved, sorted by FACS and used in multiple platforms. As proof of concept, ACME-cells are combined with SPLiT-seq (Rosenberg et al., 2018), obtaining over 32,000 cell transcriptomes and profiling two comprehensive single-cell atlases for the planarian species Schmidtea mediterranea and Dugesia japonica. Later, I use the same pipeline in a single-cell RNA interference study, in S. mediterranea, to describe and quantify the effects of the hnf4 knockdown at tissue-resolution. Our results reveal cellular and genetic changes in the gut and parenchymal populations of knockdown animals, proving that our transcriptomic approach can finely dissect tissue specific effects in knockdown conditions. In the last part of the thesis, I present the single-cell atlases of multiple planarians (Schmidtea mediterranea, Schmidtea polychroa, Dugesia japonica, Girardia tigrina and Polycelis nigra), and compare the differences between sexual and asexual strains and life-history stages of the same species. This data includes over 116,000 cell transcriptomes, and sets the bases for a future cross-species comparison that will explore the diversity of cell types and gene expression in planarians at single-cell resolution. Overall, these examples show how single-cell transcriptomics is moving toward the end of the era of descriptive cell type atlases into more complex quantitative and evolutionary studies.

Grantor dc:publisher

Oxford Brookes University

Year dc:date

2023

Author dc:creator

• García Castro, Helena

Contributors dc:contributor

• Solana, Jordi

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/rf1w-y467

OAI identifier oai:identifier

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