造血干细胞（HSCs）的下一代测序方案。逐步概述及故障排除指南。
Next-generation sequencing protocol of hematopoietic stem cells (HSCs). Step-by-step overview and troubleshooting guide.
作者信息
Jarczak Justyna, Bujko Kamila, Brzeźniakiewicz-Janus Katarzyna, Ratajczak Mariusz, Kucia Magdalena
机构信息
Laboratory of Regenerative Medicine at Medical University of Warsaw, Warsaw, Poland.
Department of Hematology, University of Zielona Gora, Multi-Specialist Hospital Gorzow Wlkp., Zielona Gora, Poland.
出版信息
PLoS One. 2025 Jan 9;20(1):e0313009. doi: 10.1371/journal.pone.0313009. eCollection 2025.
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.
在成人外周血中少量循环的极小型胚胎样干细胞（VSELs）群体（CD34⁺lin⁻CD45⁻和CD133⁺lin⁻CD45⁻）已在多种人体组织中被鉴定出来，它们与造血干细胞（HSCs）群体（CD34⁺lin⁻CD45⁺）和CD133⁺lin⁻CD45⁺共同构成了具有自我更新和多能干细胞特征的细胞库。利用先进的细胞染色和分选策略，我们分离出VSELs和HSCs群体用于大量RNA测序（RNA-Seq）分析，以比较这两种细胞群体的转录组图谱。文库是从极少数量的细胞中制备的；然而，它们的质量良好，符合测序标准。我们在此展示了一个用于VSELs和HSCs测序的逐步NGS方案，并描述了文库制备和测序过程中的故障排除方法。
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