![]() ![]() ![]() 8), as well as to the investigation of maternal–fetal immune responses triggered by this virus 9. Using our scRNA-seq data of the placenta, we have contributed to the understanding of the maternal–fetal transmission of the coronavirus SARS-CoV-2 (ref. Recently, we engaged in deciphering the physiologic and pathologic processes in the human placenta at single-cell resolution 7. The large datasets resulting from scRNA-seq allow for a wide range of different analytical pipelines that can be used for purposes as simple as identifying cell types of interest, to complex analyses such as trajectory-based differentiation or cell–cell interactions. Another advantage of droplet-based scRNA-seq methods, such as that described in the current protocol, is the unbiased nature of cell selection that requires no operator input and thus provides the best overview of cellular composition (within the technical limits of the chosen single-cell platform). Moreover, the ability to evaluate gene expression on a per-cell basis results in greater sequencing throughput compared with traditional methods that require cell sorting of target cell populations for subsequent transcriptomic analysis 6, and in this way, the stochasticity of gene expression across different cell types can be accounted for. Compared with bulk transcriptomics, single-cell RNA-sequencing (scRNA-seq) provides unbiased evaluation of tissues as heterogeneous composites of multiple cell types with distinct gene expression profiles, rather than a combined pool of transcripts 5. However, the complex cellular interactions taking place in the placenta have challenged the field of maternal–fetal medicine to implement single-cell technologies for the deeper characterization of this essential organ. Investigations of the placenta have largely been limited to histological 3 and bulk transcriptomic 4 studies. Therefore, deciphering the processes that take place in the placenta is essential for improving the understanding of fetal development and the mechanisms of obstetrical syndromes. The placenta serves as the lungs, gut, kidneys and liver of the growing fetus 1 and acts to shield the fetus against invading microbes 2. This protocol takes ~6 h to complete from tissue collection to cryopreservation of single-cell suspensions, and an additional 2 h for thawing of cryopreserved single cells. ![]() This protocol reliably produces single-cell suspensions from the placental tissues with high yield and viability for scRNA-seq. Moreover, the single-cell suspensions generated by using this protocol are compatible with droplet-based scRNA-seq technology, such as the 10x Genomics Chromium system. The protocol can be performed by a qualified investigator with basic working knowledge of placental structure. The protocol outlines the collection of tissues from the placenta, tailored dissociation procedures for each tissue, and the cryopreservation of single-cell suspensions for multiplex sequencing library preparation. Here we describe a protocol for the preparation of high-quality single-cell suspensions from human placental tissues-namely, the basal plate, placental villi and chorioamniotic membranes. So far, no protocols have been described for preparing such suspensions from the placenta, an essential organ for fetal development and a site of maternal–fetal immune interaction. An essential prerequisite for scRNA-seq is the preparation of high-quality single-cell suspensions. Single-cell RNA-sequencing (scRNA-seq) allows the characterization of cellular composition and interactions in complex tissues. ![]()
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |