title: Illumina HiSeq 2500 paired end sequencing; Leukemogenic MLL-ENL fusions induce alternative chromatin states to drive a functionally dichotomous group of target genes
design: Leukemogenic MLL-ENL fusions induce alternative chromatin states to drive a functionally dichotomous group of target genes
library_construction_protocol: 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 Takacova, S. et al. DNA damage response and inflammatory signaling limit the MLL-ENL-induced leukemogenesis in vivo. 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). Inactivation of MLL-ENL was achieved by washing out TAM keeping other media composition constant. 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. For ChIP of the estrogen receptor fusion a two-step protocol was used. After over-night incubation with anti-ER antibodies, anti-mouse immunoglobulins were added to enhance the signal.  Antibodies and concentrations used : anti-ER (TE111.5D11) from ThermoScientific (Rockford, IL) 2.5ug per assay; anti-mouse IgG/M (H+L), ThermoScientific/Pierce 31198, 2.5ug per assay; anti-H3K79 dimethyl, AbCam 3594 polyclonal AB, 2ug; anti-RNApolII CTD serine2 phospho, ActiveMotif 61083, 10ug; anti-total RNApolII, ActiveMotif 39097, 2ug, anti-H3K4 di/trimethyl, ActiveMotif 39159 polyclonal, 3ul. ChIP Seq library construction with Illumina Kit according to the instructions of the manufacturer