Illumina HiSeq 2500 paired end sequencing; RNA sequencing comparing the effect of the introduction of RUNX1-D171N in Csf3r-d715 mice bone marrow cells
RNA sequencing comparing the effect of the introduction of RUNX1-D171N in Csf3r-d715 mice bone marrow cells
Bone marrow (BM) was isolated from femurs, tibias and sternum of the mice. Cells were harvested by crushing bones in a mortar, and the harvested marrow depleted from erythrocytes by Stem-Kit lysing solution (Beckman Coulter). The early stem and progenitor compartment was enriched using the Biotin Mouse Hematopoietic Progenitor (Stem) Enrichment Set (Thermo Fisher Scientific), according to the manufacturer's protocol and subsequently used for in vitro experiments. 1e5 lineage depleted bone marrow cells per ml were plated after transduction into LODISH stem cell expansion medium. LODISH medium consisted of CellGro GMP serum-free stem cell growth medium (SCGM, CellGenix, Freiburg, Germany) supplemented with 10 ng/ml each of SCF, thrombopoietin (TPO, Preprotech), insuline-like growth factor (IGF, R&D systems), fibroblast growth factor (FGF, Preprotech) and with or without 50 ng/ml CSF3. Angiopoietin was harvested as conditioned medium of transfected HEK293T cells, and added to the culture medium in a 1:5 dilution.  LK cells were directly sorted in TRIzol (Thermo Fisher Scientific) and RNA was isolated according to the manufacturer's protocol, with the addition of GenEluteTM−LPA(Sigma-Aldrich). cDNA was generated with the SMARTer Ultra Low Input RNA kit for sequencing (version 3, Clontech).