Illumina HiSeq 2000 sequencing; A toggle-switch between miR-142 and LIF-signaling creates heterogeneity among mouse ES cells
A toggle-switch between miR-142 and LIF-signaling creates heterogeneity among mouse ES cells
Mouse ESCs were maintained without feeders in 'LIF+serum' medium (DMEM (high glucose, no glutamine, with sodium bicarbonate, Invitrogen) supplemented with 15% ES-qualified EmbryoMax Fetal Calf Serum (Millipore), 10 ng/ml murine LIF (EMBL Protein Expression and Purification Core Facility), 1x Non-Essential Amino Acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 ug/ml strep- tomycin, 0.1 mM 2-mercaptoethanol (all Invitrogen) on culture dishes (Nunc) coated with 0.1% gelatin (Sigma) solution and cultured at 37C with 5% CO2. Medium was changed daily and cells were passaged every other day with 0.05% Trypsin-EDTA (Invitrogen) at a passaging ratio of 1/6- 1/12. N2B27 medium was prepared from a 1:1 mixture of DMEM/F12 (without HEPES, with L-glutamine) and neurobasal medium with 0.5x B-27 (without vitamin A) and 0.5x N-2 supplements, 100 U/ml penicillin and 100 ug/ml streptomycin, 0.25 mM L-glutamine, 0.1 mM 2-mercaptoethanol (all Invitrogen), 10 ug/ml BSA fraction V and 10 ug/ml human recombinant insulin (both Sigma). '2i' medium was N2B27 medium supplemented with 3 uM CHIR99021 and 1uM PD0325901 (both Tocris Bioscience). mESCs were differentiated to endoderm precursors in Advanced RPMI, 100 U/ml penicillin and 100 ug/ml streptomycin (both Invitrogen), 0.2% FCS (EmbryoMax, Millipore), 1 uM IDE-1 (Tocris) following Borowiak et al. (Cell Stem Cell 2009). Barcoded miRNA libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs) following the manufacturers instructions