NextSeq 500 sequencing; Characterization of the self-organization of primitive streak and neuroectoderm fates
Cells were trypsinized, dissociated to single-cell suspension and pelleted at 1000g for 1 min. For FACS-purification, they were resuspended in D-PBS and strained through a 40 μm filter. Cells were FACS-purified according to their fluorescence levels using an Aria Fusion sorter (BD BioSciences). 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), 2 mM L-glutamine (Sigma), 1x Non-Essential Amino Acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/ml streptomycin, 0.1 mM 2-mercaptoethanol (all Invitrogen) on culture dishes (Nunc) coated with 0.1% gelatin (Sigma) solution and cultured at 37◦C with 5% CO2. mESCs were treated for 2 days with 3 μM CHIR99021 (Tocris).  For differentiation towards a primitive streak-like fate (PS-like diff), mESCs were seeded at a density of 30-200 cells per mm2 onto 0.1% gelatin coated dishes one day prior to the start of the differentiation procedure. The following day, cells were rinsed in D-PBS and switched to Advanced RPMI 1640 (ThermoFisher) supplemented with 1 μM IDE-1 (Tocris), 0.2% (v/v) ES cell qualified fetal calf serum (Millipore), 2 mM L-glutamine (Sigma). 48 hours after the onset of differentiation, medium was replaced every day.  Treatment with the RAR antagonist AGN193109 (AGN, 200 nM) was initiated at day 3 of PS-like differentiation.  For retinoic acid-mediated differentiation to neural progenitors, mESCs were seeded at a density of 100-200 cells per mm2 onto 0.1% gelatin coated dishes one day prior to the start of the differentiation procedure. The following day, cells were washed in D-PBS and switched to N2B27 medium (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 (with vitamin A) and 0.5x N-2 supplements, 0.25mM L-glutamine, 0.1mM 2-mercaptoethanol (all Invitrogen), 10μg/ml BSA fraction V and 10μg/ml human recombinant insulin (both Sigma)). all-trans-Retinoic acid (Sigma) was added at 1 μM to the differentiation medium 24 h after the start of the differentiation procedure. Medium was replaced every other day. For differentiation to neural progenitors mediated by TGFβ signaling inhibition (SB43 diff), mESCs were seeded at a density of 30 cells per mm2 onto 0.1% gelatin coated dishes one day prior to the start of the differentiation procedure. The following day, cells were rinsed in D-PBS and switched to Advanced RPMI 1640 (ThermoFisher) supplemented with 0.2% (v/v) ES cell qualified fetal calf serum (Millipore), 2mM L-glutamine (Sigma) and 10μM SB431542 (Tocris). 3μM CHIR99021 (Tocris) was added to the medium from day 3 onwards. 48 hours after the onset of differentiation, medium was replaced every day. For differentiation to neural progenitors mediated by Wnt signaling inhibition (XAV diff), mESCs were seeded at a density of 30 cells per mm2 onto 0.1% gelatin coated dishes one day prior to the start of the differentiation procedure. The following day, cells were rinsed in D-PBS and switched to Advanced RPMI 1640 (ThermoFisher) supplemented with 0.2% (v/v) ES cell qualified fetal calf serum (Millipore), 2mM L-glutamine (Sigma) and 1 μM IDE-1 and 1 μM XAV939 (both Tocris). 48 hours after the onset of differentiation, medium was replaced every day.  For Activin A- and CHIR- mediated differentiation, mESCs were seeded at a density of 30 cells per mm2 onto 0.1% gelatin coated dishes one day prior to the start of the differentiation procedure. The following day, cells were rinsed in D-PBS and switched to Advanced RPMI 1640 (ThermoFisher) supplemented with 0.2% (v/v) fetal calf serum (Millipore), 2 mM L-glutamine (Sigma) and 3 μM CHIR99021 (Tocris) or 50 ng/ml Activin A (Peprotech). 48 hours after the onset of differentiation, medium was replaced every day. RNA was extracted from pellets of trypsinized or FACS-purified cells using the MirVana kit (Ambion) following the instructions provided by the manufacturer. mRNA libraries were prepared using TruSeq RNA Sample Preparation (Illumina) following the manufacturer's instructions.