NextSeq 500 paired end sequencing; CUT&RUN-seq of H3K4me3, H3K27me3 and H3K27ac in mll2[Y2602A] mESC
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 37oC 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. DNA library was generated with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB #E7645S).