title: Illumina HiSeq 2500 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 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