Hoxb8 immortalized cell lines. Briefly, Ficoll separated bone marrow cells from 3 R26-CreERT2Srsf2P95H/+ and 3 R26-CreERT2Srsf2+/+ mice, were stimulated for 48 hours in complete Iscove modified Dulbecco medium (IMDM [Sigma Aldrich] containing 20% fetal bovine serum [FBS, Assay Matrix], 1% penicillin/streptomycin [Gibco], 1% glutamine [Gibco]) supplemented with recombinant mouse stem cell factor (50 ng/mL, PeproTech), recombinant mouse interleukin-3 (10 ng/mL, PeproTech), and recombinant human interleukin-6 (10 ng/mL, Amgen). After stimulation, 1 × 106 cells were spin-infected with Hoxb8 retrovirus20 (link) (Hoxb8 plasmids were generously provided by Mark Kamps, University of California San Diego) and polybrene at 1100g for 90 minutes. After 48 hours, cells were then passaged into complete IMDM containing 1% granulocyte-macrophage colony-stimulating factor (GM-CSF) conditioned medium (from BHK-HM5 cell conditioned medium).
Cas9 expressing cell lines. Hoxb8 immortalized cell lines were infected with Cas9-blasticidin lentivirus (lentiCas9-Blast was a gift from Feng Zhang; Addgene plasmid #52962)21 (link) by spin infection at 1100g for 90 minutes. The infected cells were then cultured in IMDM-Cas9 medium (IMDM, 10% FBS, 1% GM-CSF, and 3 µg/mL blasticidin) for 2 weeks to select for a Cas9-expressing population.
Cas9 expressing cell lines. Hoxb8 immortalized cell lines were infected with Cas9-blasticidin lentivirus (lentiCas9-Blast was a gift from Feng Zhang; Addgene plasmid #52962)21 (link) by spin infection at 1100g for 90 minutes. The infected cells were then cultured in IMDM-Cas9 medium (IMDM, 10% FBS, 1% GM-CSF, and 3 µg/mL blasticidin) for 2 weeks to select for a Cas9-expressing population.