Next-generation sequencing protocol of hematopoietic stem cells (HSCs). Step-by-step overview and troubleshooting guide.
basic_science · Level V
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- Record sourced from PubMed, PMID 39787063.
- Also identified by DOI 10.1371/journal.pone.0313009 and PMC identifier 11717189.
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Abstract
Populations of very small embryonic-like stem cells (VSELs) (CD34+lin-CD45- and CD133+lin-CD45-), circulating in the peripheral blood of adults in small numbers, have been identified in several human tissues and together with the populations of hematopoietic stem cells (HSCs) (CD34+lin-CD45+) and CD133+lin-CD45+constitute a pool of cells with self-renewal and pluripotent stem cell characteristics. Using advanced cell staining and sorting strategies, we isolated populations of VSELs and HSCs for bulk RNA-Seq analysis to compare the transcriptomic profiles of both cell populations. Libraries were prepared from an extremely small number of cells; however, their good quality was preserved, and they met the criteria for sequencing. We present here a step-by-step NGS protocol for sequencing VSELs and HSC with a description of troubleshooting during library preparation and sequencing.
Medical subject headings
- Hematopoietic Stem Cells
- Hematopoietic Stem Cells/metabolism
- Hematopoietic Stem Cells/cytology
- Humans
- High-Throughput Nucleotide Sequencing
- High-Throughput Nucleotide Sequencing/methods