Illumina HiSeq 2500 paired end sequencing; DNA sequencing comparing different stages of leukemic progression in a mouse model of severe congenital neutropenia (SCN)
DNA sequencing comparing different stages of leukemic progression in a mouse model of severe congenital neutropenia (SCN)
Upon sacrifice, bone marrow was isolated from femurs and tibias of the mice. Cells were harvested by flushing the bones and filtered. Subsequently, GFP+ Csf3r-d715/RUNX1-D171N cells were sorted for sequencing. 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. 100 ng genomic DNA was fragmented using enzymatic fragmentation. Sample libraries were constructed following the SeqCap EZ HyperPlusCap workflow User's Guide version 1.0 (Roche). Unique, dual index adapters (Integrated DNA technologies) were used for ligation. After ligation of adapters and an amplification step, exome target sequences were captured using in-solution oligonucleotide baits (SeqCap EZ Developer Library mm9_exome_L2R_D02).