Short-read and long-read RNA sequencing of mouse hematopoietic stem cells at bulk and single-cell levels

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

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(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.

Zheng X, Zhang D, Xu M, Zeng W, Zhou R, Zhang Y, Tang C, Chen L, Chen L, Lin JW. (2021) Short-read and long-read RNA sequencing of mouse hematopoietic stem cells at bulk and single-cell levels. Sci Data 8(1):309. [article]

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