title: GSM4259841: Runx1Evi1_plusDox_Evi1_ChIPseq; Mus musculus; ChIP-Seq
library_construction_protocol: KIT+ HP cells were purified as indicated above, then crosslinked for 45 minutes at room temperature in PBS with 0.83 mg/ml Di(N-succinimidyl) glutarate (Sigma), followed by a second crosslink for 10 minutes at room temperature in PBS with 1% formaldehyde (Pierce) which was quenched by adding 1/10th volume of 2M glycine. Nuclei were prepared as ref then sonicated for 8 cycles of 30s on/30s off using a Picoruptor (Diagenode), in 25 mM Tris 1 M pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% TritonX-100 and 0.5% SDS. The chromatin fragments were diluted with 2 volumes of buffer containing 25 mM Tris pH 8.0, 150mM NaCl, 2 mM EDTA pH 8.0, 1% TritonX-100, 7.5% glycerol. Immunoprecipitation was carried out overnight at 4°C using 2 µg of RUNX1 (ab23980, Abcam) or EVI1 (2593, Cell Signalling Technologies) antibody coupled to 15 µg Dynabeads Protein G (Invitrogen) per 2 x 106 cells. The beads were then washed using low salt, high salt, LiCl and TE/NaCl buffers and eluted, the resulting material was incubated overnight with 0.25 mg/ml proteinase K. DNA from 2-3 immunoprecipitations was pooled and extracted using Ampure beads (Beckman Coulter) and ChIP quality was assessed using qRT-PCR. ChIP libraries were generated and sequenced as for DNase I using the KAPA hyper prep kit, except libraries were size selected to obtain fragments between 150 and 450 bp.
attributes.tag: GEO Accession
attributes.value: GSM4259841