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Single-Cell Analysis of Hematopoietic Stem Cells.

Katherine H M Sturgess1, Fernando J Calero-Nieto1, Berthold Göttgens1

  • 1Department of Haematology, Jeffrey Cheah Biomedical Centre, Puddicombe Way, Wellcome - MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.

Methods in Molecular Biology (Clifton, N.J.)
|May 31, 2021
PubMed
Summary

Single-cell RNA sequencing (scRNA-Seq) offers deep insights into hematopoiesis. This chapter details protocols for mouse bone marrow analysis using plate-based and droplet-based methods for comprehensive cell profiling.

Keywords:
Bone marrowGene regulatory networksHematopoiesisHematopoietic stem cellHematopoietic stem/progenitor cellSmart-Seq2TranscriptomemcSCRB-SeqmcSmart-Seq2scRNA-Seq

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Area of Science:

  • Hematopoiesis research
  • Single-cell genomics
  • Bioinformatics

Background:

  • Single-cell RNA sequencing (scRNA-Seq) has transformed hematopoiesis studies.
  • Transcriptomic data reveals novel cell populations and lineage decisions.
  • scRNA-Seq aids in understanding early stages of malignant transformation.

Purpose of the Study:

  • To provide a comprehensive protocol for scRNA-Seq analysis of mouse bone marrow.
  • To detail both plate-based (low throughput) and droplet-based (high throughput) methods.
  • To enable functional analysis of gene regulatory networks in hematopoietic cells.

Main Methods:

  • Sample preparation and antibody panel for flow cytometric purification of hematopoietic progenitors.
  • Plate-based protocol (Smart-Seq2/mcSCRB-Seq hybrid) optimized for automation.
  • Droplet-based method (e.g., 10× Genomics) utilizing manufacturer's guidelines.

Main Results:

  • Detailed protocols for scRNA-Seq of mouse bone marrow are presented.
  • Both low-throughput and high-throughput methods are described with specific steps.
  • The plate-based method yields a sequencing-ready library in 4 days; droplet-based in 3 days.

Conclusions:

  • These scRNA-Seq protocols provide a robust framework for detailed hematopoiesis research.
  • The methods facilitate identification of novel cell types and regulatory pathways.
  • This work supports functional studies and understanding of hematopoietic malignancies.