Zebrafish Thrombocyte Transcriptome Analysis and Functional Genomics.
Bioinformatics
GFP+ thrombocytes
PANTHER
Piggyback knockdown
Primer 3
RFP+ thrombocytes
Single-cell RNA-Seq
Thrombocyte
Transcriptome
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2024
2024
Historique:
medline:
28
7
2024
pubmed:
28
7
2024
entrez:
27
7
2024
Statut:
ppublish
Résumé
Our laboratory is interested in investigating the maturation process of zebrafish thrombocytes, which are functional equivalents to human platelets. We have adopted the zebrafish model to gain insights into mammalian platelet production, or thrombopoiesis. Notably, zebrafish exhibit two distinct populations of thrombocytes in their circulating blood: young and mature thrombocytes. This observation is intriguing because maturation appears to occur in circulation, yet the precise mechanisms governing this maturation remain elusive. Our goal is to understand the mechanisms underlying thrombocyte maturation by conducting single-cell RNA sequencing (scRNA-Seq) on young and mature thrombocytes, analyzing these transcriptomes to identify genes specific to each thrombocyte population, and elucidating the role of these genes in the maturation process, by quantifying thrombocyte numbers after the piggyback knockdown of each of these genes. In this chapter, we present a comprehensive, step-by-step protocol detailing the multifaceted methodology involved in understanding thrombocyte maturation, which encompasses the collection of zebrafish blood, the separation of young and mature thrombocytes using flow cytometry, scRNA-Seq analysis of these distinct thrombocyte populations, identification of genes specific to young and mature thrombocytes, and subsequent validation through gene knockdown techniques.
Identifiants
pubmed: 39068363
doi: 10.1007/978-1-0716-3886-6_10
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
193-201Informations de copyright
© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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