RNA-seq of mus musculus: adult female bone marrow
Leukemia cells isolated from bone marrow of Evi1-overexpressing leukemia mice were crosslinked with 1% formaldehyde at room temperature for 10 minutes, and the reaction was stopped by the addition of 80 mM glycine. The fixed cells were rinsed three times with cold phosphate-buffered saline (PBS) and stored at -80 . Cells were washed in lysis buffer resuspended in micrococcal nuclease (MNase) buffer and rocked on ice for 10 minutes. After centrifugation, each pellet was resuspended in MNase buffer with 5 mM CaCl2, and subjected to MNase digestion at 10000 U/mL (New England Biolabs Japan, Japan). The digestion was stopped by adding EDTA after 20 minutes of incubation at 37  and the reaction mixture was diluted with buffer-L, followed by ultrasonication. After sonication, 40 l of Dynabeads Protein G mixed with 5 g of each antibody for three hours were added to the lysates and incubated by rotating overnight, washed for five times using Magnetic stand with wash buffer. Bound DNA fragments were eluted and quantified by next generation sequencing performed by Illumina Hiseq. The antibody used in ChIP assays was monoclonal Anti-FLAG M2 antibody (F3165, Sigma-Aldrich).