ChIP-Seq of Mus Musculus: young male/female spleen
Nave CD8+ T cells from Kmt2dWT, Kmt2d-/- mice were sorted by FACS and stimulated with anti-CD3/CD28 in vitro for 24 hours. Human CD4 nave cells were sorted for spike in control. Cells were digested in digestion buffer (50mM Tris-HCl, 1mM CaCl2, 0.2% Triton X-100, 5mM sodium butylate with proteinase inhibitor cocktail and 0.5mM PMSF). Digested human CD4N cells were spiked in to mouse cell digestions at the ratio of 10%. 0.2U MNase was added per million cells and incubated at 37C for 40 minutes and sonicated (Diagenode) with a power setting of 5 for 4 repeats of 30 seconds followed by 30 seconds break between each repeat in ice-cold water to obtain the average size between 100-300 bps. The sonicated products were dialyzed with 10000 MWCO Cassette G2 (Thermo-Fisher) in RIPA buffer for 2 hours at 4C, and then incubated with Dynabeads Protein G (Thermo-Fisher) coated with 4mg of anti-H3K4me1 antibody or non-specific IgG (Millipore) for overnight. The precipitated beads were washed with RIPA, LiCl and TE buffer sequentially, and recovered overnight at 65C with TE supplemented with 10% SDS and proteinase K (MBiotech). The ChIP DNA was purified with phenol/chloroform (Invitrogen) and ethanol precipitation.