Illumina HiSeq 2500 paired end sequencing; RNA sequencing comparing different stages of leukemic progression in a mouse model of severe congenital neutropenia (SCN)
RNA sequencing comparing different stages of leukemic progression in a mouse model of severe congenital neutropenia (SCN)
Peripheral blood (PB) was obtained by cheek puncture. Upon sacrifice, BM was isolated from femurs and tibias of the mice. Cells were harvested by flushing the bones and filtered. Subsequently, lineage- c-Kit+ (LK) cells were FACS sorted. Mice were injected subcutaneously three times a week with 100 µg/kg CSF3 (Filgrastrim, Zarzio) or vehicle control (PBS). Treatment was started five weeks after transplantation in the primary recipients. The secondary recipients started CSF3 treatment four weeks after transplantation, while the tertiary transplant recipients received CSF3 or PBS treatment two weeks after transplantation. The tertiary recipients received a higher dose of CSF3 (200 μg/kg). From the start of the experiment, all mice received 50 μg/ml of Ciprofloxacin in the drinking water. 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 4, Clontech).