E14.5 embryos were dissected from uterus; head, limbs and internal organs were removed. Tissue was disaggregated using an 18-gauge syringe and brought to single cell suspension with trypsin incubation at 37°C. Cells were then plated onto 100 mm tissue culture plates, passaged upon confluency and maintained in Dulbecco's modified Eagle medium (DMEM-Sigma) with 10% fetal bovine serum (FBS-Gibco) and 1X Pen/Strep antibiotics (Sigma). SV40 T immortalized MEFs (iMEFs) were derived from primary Hat1+/+ and Hat1−/− embryonic day 13.5 embryos. To establish iMEFs, early passage cells were transformed with SV-40 T antigen containing plasmid pBSSVD2005 (ADDGENE, Cambridge, MA). Early passage cells were seeded at 25% confluency in six-well plates and transfected with 2 ug of expression vector using Fugene reagent (Roche). Cells were harvested and seeded into 100 mm dishes after 48 h of transfection. The cells were split at 1 in 10 dilutions until passage 5.
For the SILAC experiments, cells were grown in SILAC DMEM media (ThermoFisher Scientific) and supplemented with 10% dialyzed fetal bovine serum (ThermoFisher Scientific), antibiotics and isotopically labeled lysine and arginine (Cambridge Isotope laboratories). Hat1−/− cells were grown under these conditions for at least four passages to guarantee the full incorporation of the labeled amino acids.
For the SILAC experiments, cells were grown in SILAC DMEM media (ThermoFisher Scientific) and supplemented with 10% dialyzed fetal bovine serum (ThermoFisher Scientific), antibiotics and isotopically labeled lysine and arginine (Cambridge Isotope laboratories). Hat1−/− cells were grown under these conditions for at least four passages to guarantee the full incorporation of the labeled amino acids.