Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and JAK2-V617F expressing hematopoietic stem and progenitor subsets

Transcriptomics analysis was performed on FACS purified HSPC subsets from SclCre;V617F mice and WT mice bone marrow. The goal of this study is to identify the molecular signatures that are specific to the mutant JAK2 expressing HSPC subsets. We found that mutant JAK2 activation caused dysregulated expression of large numbers of genes in primitive HSPC subsets. Furthermore, this analysis revealed molecular identity and developmental proximity of HSC CD41+/- cells within the HSPC hierarchy. To activate JAK2-V617F mutation, SclCre;V617F (SF1) transgenic mice and Wildtype (WT) were treated with tamoxifen for 5 consequetive days. 12 weeks post-tamoxifen injections, titbia and femur from both legs were havested and curshed in a mortor and pistle to extract total bone marrow cells. Total bone marrow cells were subjected to ACK lysis to remove erythrocytes. Cells were then surface stained with the antibodies to identify hematopoietic stem and progenitor subsets. Following subsets were isolated using FACS Aria3: Long-term Hematopoietic Stem Cells (LT-HSC; Lineage negative/cKit+/Sca-1+/CD48-/CD150+); CD41+ HSCs Lineage negative/cKit+/Sca-1+/CD48-/CD150+CD41+); CD41-HSCs (Lineage negative/cKit+/Sca-1+/CD48-/CD150+ CD41-); Megakaryocyte progenitors (MkPs: Lineage negative/cKit+/Sca-1-/CD150+CD41+). Cells were sorted directly into eppendorf tubes containing RNA lysis buffer. Total RNA was prepared using Acturus PicoPure RNA preparation kit (Applied Biosystems). RNA samples with RIN value >8 were used for library preparation using Illumina RNA Seq library preparation kit.