GSM1671299: Rep1_flox_K4me1; Mus musculus; ChIP-Seq
Immature myeloid cells (iGMP cells; GFP+, CD11b-, Gr1-, CD16/32+) were sorted from cultures. Genomic DNA was isolated using the AllPrep DNA/RNA micro kit (Qiagen). Duplicate cultures of Tet2fl/fl;AE or Tet2-/-:AE cells grown in vitro for 10 passages after Tet2 disruption were fixed for 10 minutes in 1% formaldehyde dissolved in PBS and the reaction was quenched by addition of glycine to a final concentration of 0.125M. The crosslinked cells were harvested in SDS lysis buffer (100 mM NaCl, 50mM Tris-HCl (pH8.1), 5mM EDTA (pH 8.0), 0.2% NaN3, 0.5% SDS) and nuclei were pelleted and resuspended in IP buffer (100mM Tris-HCl (pH 8.6), 100mM NaCl, 5mM EDTA (pH 8.0), 0.2% NaN3, 5% Triton X-100, 0.3% SDS). The obtained DNA was then sheared to an average size of 150-300bp (Bioruptor, Diagenode) and 40ug of solubilized chromatin was immunoprecipitated with Protein A Sepharose beads (GE healthcare) after overnight incubation with the respective antibodies. Finally, adaptor-ligated libraries were generated from 10ng of precipitated DNA using the NEBNext® DNA library Sample Prep Master kit (New England Biolabs), PCR-amplified (15x PCR cycles) using indexed multiplex primers for illumina sequencing (New England Biolabs), and sequenced on a HiSeq2000 using 50bp single-end sequencing at the National High-Throughput Sequencing Centre (University of Copenhagen).
GEO Accession
GSM1671299