mll2wt_H3K27ac_rep3
Mus musculus
Protocols: Naive murine embryonic stem cells (mESC) were derived freshly (FVB or mixed 129/B6) and were routinely cultured on gelatin-coated plates in t2i/L media: NDiff (N2B27) (Takara #y40002) supplemented with titrated 2i (0.2 �M PD0325901 and 3 �M CHIR 99021), 1,000 U/ml leukaemia inhibitory factor (LIF), 1% FBS, 1% penicillin streptomycin and maintained in humidified atmosphere at 37C and 5% CO2. Cells were passaged every 2 days by dissociation with TrypLE and medium was changed daily. This protocol has been optimised from Skene and Henikoff, Elife, 2017 to detect interaction between DNA and histone modifications. Briefly, fresh cells are harvested, permeabilised, bound to concanavallin magnetic beads and incubated with the antibody for the modification of interest. Samples were then incubated with pA-MNase to cut the DNA side of the bound antibody and the DNA fragments were eluted and subjected to size selection. Finally DNA library was generated with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S).