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Single-Cell Analysis of Hematopoietic Stem Cells.

Accepted version
Peer-reviewed

Type

Article

Change log

Authors

Sturgess, Katherine HM 
Calero-Nieto, Fernando J 
Göttgens, Berthold 
Wilson, Nicola K 

Abstract

The study of hematopoiesis has been revolutionized in recent years by the application of single-cell RNA sequencing technologies. The technique coupled with rapidly developing bioinformatic analysis has provided great insight into the cell type compositions of many populations previously defined by their cell surface phenotype. Moreover, transcriptomic information enables the identification of individual molecules and pathways which define novel cell populations and their transitions including cell lineage decisions. Combining single-cell transcriptional profiling with molecular perturbations allows functional analysis of individual factors in gene regulatory networks and better understanding of the earliest stages of malignant transformation. In this chapter we describe a comprehensive protocol for scRNA-Seq analysis of the mouse bone marrow, using both plate-based (low throughput) and droplet-based (high throughput) methods. The protocol includes instructions for sample preparation, an antibody panel for flow cytometric purification of hematopoietic progenitors with index sorting for plate-based analysis or in bulk for droplet-based methods. The plate-based protocol described in this chapter is a combination of the Smart-Seq2 and mcSCRB-Seq protocols, optimized in our laboratory. It utilizes off-the-shelf reagents for cDNA preparation, is amenable to automation using a liquid handler, and takes 4 days from preparation of the cells for sorting to producing a sequencing-ready library. The droplet-based method (using for instance the 10× Genomics platform) relies on the manufacturer's user guide and commercial reagents, and takes 3 days from isolation of the cells to the production of a library ready for sequencing.

Description

Keywords

Bone marrow, Gene regulatory networks, Hematopoiesis, Hematopoietic stem cell, Hematopoietic stem/progenitor cell, Smart-Seq2, Transcriptome, mcSCRB-Seq, mcSmart-Seq2, scRNA-Seq, Animals, Cell Lineage, Cell Separation, Flow Cytometry, Gene Expression Profiling, Gene Library, Gene Regulatory Networks, Hematopoiesis, Hematopoietic Stem Cells, Mice, Phenotype, RNA-Seq, Single-Cell Analysis, Transcriptome

Journal Title

Methods Mol Biol

Conference Name

Journal ISSN

1064-3745
1940-6029

Volume Title

2308

Publisher

Springer US

Rights

All rights reserved
Sponsorship
Wellcome Trust (206328/Z/17/Z)
Bloodwise (18002)
Medical Research Council (MR/S036113/1)
Wellcome Trust (203151/Z/16/Z)
Medical Research Council (MR/M008975/1)
This is a methods chapter which didn't include a funding statement. We can request the publisher if this can be added during proof stage.