Esc grade fbs

Feeder dependent female WT and Ptbp1^−/− BL6 mouse ES cells were expanded on inactive male murine embryonic fibroblast (MEF) feeders on 0.1% gelatinized tissue culture plates in DMEM media (Gibco cat. no. 10313039) supplemented with 15% ESC grade FBS (Gibco cat. no. 10439024), 1% nucleosides (EMD Millipore cat. no. ES008D), 1% Glutamax (Gibco cat. no. 35050061), 0.1 mM β-mercaptoethanol (Acros Organics cat. no. 125472500), and 1000 U/ml mLIF (EMD Millipore cat. no. ESG1106). Feeder independent female WT and Ptbp1^−/− BL6 mouse ES cells, and F1 2-1 mouse ES cells were expanded on 0.1% gelatinized tissue culture plates in 2i culture media containing 50% Neurobasal (Gibco cat. no. 21103049) and 50% DMEM/F12 (Gibco cat. no. 11320082) supplemented with 1% B27 + RA (Gibco cat. no. 17504044), 1% N2 Supplement (R&D System cat.no. AR009), 1% Glutamax, 7.4 mM B27 Fraction V (Gibco cat. no. 15260037), 1% Penicillin-Streptomycin (GE Healthcare Life Sciences cat. no. SV30010), 3 μM CHIR99021 (Sigma cat. no. SML1046), 1 μM PD0325901 (Selleckchem cat. no. S1036), 150 μM 1-thioglycerol (Sigma cat. no. M6145) and 1000 U/ml mLIF (Gemini Bio-Products cat. no. 400–495). To assist initial attachment, 2i medium was supplemented with 2% ESC grade FBS for the first 24 h before switching to serum free.