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 22. 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. Generation and purification of nascent RNA To determine RNA synthesis rates, newly transcribed RNA was labeled by addition of 100uM 4-thio-uridine (Sigma, Taufkirchen, Germany) directly to the cell culture medium. After 1h total RNA was isolated by RNeasy spin column purification according to the instructions of the manufacturer (Quiagen, Hilden, Germany). 100 ug of RNA were biotinylated with 200ug/ml EZ-Link HPDP Biotin (Pierce-Thermo, Rockford, IL) in 1ml of 10mM Tris-HCl pH7.4, 1mM EDTA. Biotinylated nucleic acid was bound to uMACS streptavidin magnetic beads and washed as recommended by the manufacturer (Miltenyi, Bergisch-Gladbach, Germany). Elution of retained material was done with 100mM DTT. After a final purification on RNeasy columns nascent RNA was subjected to standard reverse transcription and qPCR. random primed library using nascent RNA as input
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2016-04-01T17:05:11Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-09T01:17:01Z
attributes.tag: External Id
attributes.value: SAMEA3403311
attributes.tag: INSDC center name
attributes.value: FAU Erlangen, Division of Genetics
attributes.tag: INSDC first public
attributes.value: 2016-04-01T17:05:11Z
attributes.tag: INSDC last update
attributes.value: 2018-03-09T01:17:01Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3591:Sample 2
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: CD117-positive primary hematopoietic precursor cell
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 Mll locus 22
attributes.tag: growth condition
attributes.value: cell suspension culture
attributes.tag: sample name
attributes.value: E-MTAB-3591:Sample 2
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: Meer
attributes.tag: time
attributes.value: 8 hour