Next-generation sequencing protocol of hematopoietic stem cells (HSCs). Step-by-step overview and troubleshooting guide.

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.
Competing Interests: The authors have declared that no competing interests exist.
(Copyright: © 2025 Jarczak et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)