title: ENL_smpl1
organism: Mus musculus
description: Protocols: For generation of Meer cells primary hematopoietic precursor cells were isolated from the bone marrow of Meer mice that carry a knock-in of an ENL-ER (mutant estrogen receptor ligand binding domain) portion into the mouse Mll locus. (Lit: Takacova, S. et al. Cancer Cell 21, 517-31, 2012). CD117 (c-kit) positive cells were isolated by magnetic enrichment according to the instructions of the manufacturer (CD117 enrichment kit, Miltenyi Biotech, Bergisch-Gladbach, Germany) and initially plated in methocel (MethoCult M3234, StemCellTechnologies, Koln, Germany) to purge residual stroma cells that cannot grow in semisolid media. After two rounds of growth in methylcellulose cells were expanded in RPMI1640 supplemented with 10% FCS. All media contained 50ng/ml recombinant murine (rm) SCF, 10ng/ml of rm-IL3, IL6, GM-CSF (cytokines were bought from Miltenyi Biotech, Bergisch-Gladbach, Germany) and for activation of the estrogen receptor 100nM 4-hydroxytamoxifen (TAM, Sigma, Taufkirchen, Germany). ENL and ENLins cells were derived from CD117 selected primary bone marrow cells isolated from wild type Balb/C mice transduced with ENL and ENLins retroviral constructs and cultivated as described for Meer cells. ChIP was done exactly as described in (Milne, T.A., Zhao, K. & Hess, J.L. Chromatin immunoprecipitation (ChIP) for analysis of histone modifications and chromatin-associated proteins. Methods Mol Biol 538, 409-23 (2009) with the exception that protein G-coupled paramagnetic beads from CellSignalingTechnologies (CST/NEB, Frankfurt, Germany) were used as precipitating agent. Antibodies and concentrations used : anti-H2AUb, Cell Signaling Technologies (#8240) 10µl; anti-H2BUb, Cell Signaling Technologies (#5546) 5µl, anti-PAF1, 5µl AbCam rabbit polyconal (#137519) + 5µl Cell Signaling Technologies (#12883) per 5x106 cells, anti-MLLT1(ENL) 15µl Bethyl rabbit polyclonal (#A302-268A) per 5x106 cells. ChIP Seq library construction with Illumina Kit according to the instructions of the manufacturer