mll2wt_EpiLC_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. To induce epiblast-like cell (EpiLC) differentiation, naive mouse ESCs were seeded on fibronectin-coated plates in NDiff (N2B27) (Takara #y40002) supplemented with 20 ng/ml ActivinA (PeproTech #120-14P), 12 ng/ml bFGF (PeproTech #450-33), 1% knockout serum replacement (Thermo Fisher #10828010) and 1% penicillin streptomycin. Cell were harvested ~40 hours after EpiLC induction. RNA-sequencing of polyA RNA from mouse Embryonic Stem Cells. Total RNA was isolated using the Monarch Total RNA Miniprep Kit (NEB #T2010S), including gDNA removal step and on-column DNase I digestion. Total RNA was quantified using Qubit II and quality assessed with high-sensitivity RNA Screen Tape (Agilent 5067-5579). Stranded mRNA-seq libraries were prepared with the NEBNext RNA Ultra II kit (input: 350 ng of total RNA) following the manufacturer's instructions.