Expression profiling of hematopoietic stem cells/progenitors from young (3 months) and old (12 months) homozygous Jak2 R1063H mice and wild-type controls
Expression profiling of hematopoietic stem cells/progenitors from young (3 months) and old (12 months) homozygous Jak2 R1063H mice and wild-type controls
Mice were sacrificed by cervical dislocation, femurs and tibias were isolated, and crunched using a pestle and mortar. Sorting of bone marrow hematopoietic stem cells and progenitors was performed by two subsequent steps. First, the Lin+ fraction of the BM cells was labeled using biotinylated lineage markers CD45/B220 (RA3-6B2), CD3 (145-2c11), Ter119 (TER-119), Gr1 (RB6-8C5), and CD11b (M1/70). These cells were then incubated with anti-biotin magnetic beads (Miltenyi Biotec) and were isolated using MACS separator. Second, the Lin- fraction of the BM was labeled with the c-Kit PE (2B8), Sca-1 APC (E13-161.7), CD48 FITC (HM48-1), CD150 Pe-Cy7 (TC15-12F12.2) antibodies and with streptavidin-eFluor450. Cell suspensions were stained with Hoechst 33258 to exclude dead cells and HSCs were sorted using Influx instrument (BD Biosciences). Total RNA was isolated using RNeasy Micro Kit (Qiagen) as described in the manufacturer's protocol. Libraries were prepared with a SMARTer® Stranded Total RNA-Seq – Pico Input Mammalian library preparation kit v2 (Takara).