title: Kmt2d ChIP-Seq of Mus Musculus: young male/female spleen
design: Nave CD8+ T cells from Kmt2d WT and Kmt2d KO mice were sorted by FACS and stimulated with aCD3/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, 5 mM sodium butylate with proteinase inhibitor cocktail and 0.5 mM PMSF). Digested human CD4N cells were spiked into mouse cell digestions at the ratio of 10%. 0.2U MNase (ThermoFisher) 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. For ChIP-Seq of Kmt2d, sonicated chromatin was incubated with Dynabeads Protein A coated with 8 mg of anti-Kmt2d/Mll4 antibody (8) at 4C overnight. Beads were washed with RIPA, RIPA with 300mM NaCl, LiCl buffer and PBS sequentially, and recovered overnight at 65C with TE supplemented with 10% SDS and proteinase K. ChIPed DNA was purified using QIAquick PCR purification kit (Qiagen). Sequencing libraries were constructed using NEBNext Ultra II DNA Library Prep kit following manufacturers instruction. AMPure XP magnetic beads (Beckman Coulter) were used for size selection. PCR amplification of libraries was done for 15 cycles.