Hematopoietic stem cells (HSCs) lie at the top of the differentiation hierarchy. Although HSC and their immediate downstream, multipotent progenitors (MPP) have full multilineage differentiation capacity, only long-term (LT-) HSC has the capacity of long-term self-renewal. The heterogeneity within the HSC population is gradually acknowledged with the development of single-cell RNA sequencing and lineage tracing technologies. Transcriptional and post-transcriptional regulations play important roles in controlling the differentiation and self-renewal capacity within HSC population.
Researchers at Sichuan University report a dataset comprising short- and long-read RNA sequencing for mouse long- and short-term HSC and MPP at bulk and single-cell levels. They demonstrate that integrating short- and long-read sequencing can facilitate the identification and quantification of known and unannotated isoforms. Thus, this dataset provides a groundwork for comprehensive and comparative studies on transcriptional diversity and heterogeneity within different HSC cell types.
Sample collection and workflow
(a) Sample collection and sequencing methods. Bone marrow cells (BMC) were isolated, and lineage negative (LIN−) cells were enriched using magnetic beads. Long-term (LT) and short-term (ST) hematopoietic stem cells (HSC) and multipotent progenitors (MPP) were sorted according to their surface makers: LT-HSC (Sca-1+c-Kit+CD34− CD135−), ST-HSC (Sca-1+ c-Kit+CD34+ CD135−) and MPP (Sca-1+ c-Kit+CD34+ CD135+). Single-cell or 100 cells (bulk P100) were sorted for library construction following the Smart-seq2 protocol. The cDNA libraries were used for short-read (Illumina Hiseq) or long-read (Nanopore or PacBio) sequencing. (b) Gating strategy for cell sorting. The main population was gated via FSC-A (forward scatter) and SSC-A (side scatter), and single cells were gated via FSC-A and FSC-H. FSC, forward scatter; SSC, side scatter; A, area; H, height.