Stemspan sfem serum free media

C/EBPα f/f Mx1-Cre + mice were intraperitoneal injected 30 µg Poly I:C (Sigma-Aldrich, Cat. No. P0913) per gram of mouse weight every alternative day for 18 days to induce C/EBPα conditional knockout. Two tibia, two femur and two pelvis were harvested from each knockout mice. Bone marrow cells were isolated from each bones and proceed to lineage depletion using direct lineage depletion kit (Milenyi Biotec, . Cells were then stained with antibody (Supplementary Table . 2) and LSK cells were isolated through cell sorting using FACSAria (BD). Sorted LSK cells were cultured in StemSpan SFEM serum free media (stemcell technologies) contains 40 ng/ml mSCF (Biolegend, Cat. No. 579702), 40 ng/ml mTPO (Biolegend, Cat. No. 593302) and 10 ng/ml IL3 (Biolegend, Cat. No. 575502) for virus transduction. 200 MOI retrovirus and 5 µg/cm 2 RetroNectin reagent (Takara, Cat. No. T100A) were used in the transduction following the manufactory protocol. 48 hours post transduction, 1 × 10 5 LSK cells per mouse were injected into lethally irradiated (1000 cGy) B6.SJLPtprca Pepcb/BoyJ (Pep Boy) congenic recipients along with 1 × 10 5 lineage depleted Pep boy bone marrow cells for radioprotection.

Retrovirus plasmids were transfected into 293T-Eco cells using Lipofectamine 2000 (Thermo scientific, Cat. No. 11668019) . 48 hours post transfection, supernatant was harvested and filtered by 0.45 µm hydrophilic PVDF filter (Foxx life science, Cat. No. 378-3415-OEM). Retro-X concentrator (Takara, Cat. No. 631456 ) was added to filtered supernatant at a ratio 1:3 and incubated overnight at 4 degree Celsius. After incubation, spindown and suspended the virus pallet in 500 µl StemSpan SFEM serum free media (stemcell technologies, Cat. No. 09650). Make a small aliquot for virus titration using qPCR titration kit (abm, Cat. No. G949). The remaining virus was put in -80 degree celsius freezer.