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 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
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2016-04-01T17:05:13Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-09T01:39:06Z
attributes.tag: External Id
attributes.value: SAMEA3443321
attributes.tag: INSDC center name
attributes.value: FAU Division of Genetics
attributes.tag: INSDC first public
attributes.value: 2016-04-01T17:05:13Z
attributes.tag: INSDC last update
attributes.value: 2018-03-09T01:39:06Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3593:Sample 1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genetic modification
attributes.value: gene knock in
attributes.tag: genotype
attributes.value: ENL-ER knock-in into Mll locus
attributes.tag: growth condition
attributes.value: cell suspension culture
attributes.tag: sample name
attributes.value: E-MTAB-3593:Sample 1
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: time
attributes.value: 0 hour