title: Multiplex CRISPR/Cas9 editing of mus musculus: Targets: Dnmt3a, Ep300, Ezh2, Gata3, Nf1; Pre-transplantation
design: c-kit+ and LSK cells were cultured in X-Vivo 15 media (Lonza) supplemented with 2% FBS, SCF (50 ng/ml), TPO (50 ng/ml), IL-3 (10 ng/ml), and IL-6 (10 ng/ml) (all from Peprotech) for 16-24 hours before electroporation. The recipient mice were lethally irradiated and transplanted with genome-edited LSK cells. Genomic DNA was extracted from donor-derived GFP+ cells sorted from peripheral blood and lysed in Lysis buffer supplemented with Proteinase K (200  g/ml, Millipore). Samples were incubated at 55 C for 30 minutes, followed by 95 C for 3 minutes to inactivate Proteinase K. The genomic regions flanking the Cas9 target site for each gene was amplified by two-rounds of PCR (amplicon size 200-300bp) to attach Illumina adapters to the amplicon. All PCR products were pooled and purified with Sera-Mag Magnetic SpeedBeads (ThermoFisher Scientific).