RNA sequencing comparing different stages of leukemic progression in a mouse model of severe congenital neutropenia (SCN)

To address how Csf3r and RUNX1 mutations in combination with CSF3 administration affect hematopoiesis in vivo, we performed serial transplantation experiments. Lineage-negative Csfr-d715 BM cell cells were lentivirally transduced with the patient specific RUNX1-D171N (RHD) mutant or an empty vector control. An additionaly acquired mutation in Cxxc4 in mouse 29 transformed the pre-leukemic condition, characterized by excess pheripheral lineage- c-kit+ (LK) myeloblasts, into acute myeloid leukemia (AML). LK blasts were FACS sorted in TriZol and RNA was isolated according to the manufacturer’s protocol. SMARTer Ultra Low Input RNA kit for sequencing (Clontech, v4 Cat# 634891) was used to generate cDNA. Sequencing libraries were generated using TruSeq Nano DNA Sample Preparation kits (Illumina, Cat# 20015964), according to the low sample protocol and paired-end sequenced on a HiSeq 2500 (Illumina).