NextSeq 500 paired end sequencing; scRNA-Seq analysis of differentiation to primitive streak fate of mouse embryonic stem cells
Cultures undergoing primitive streak-like differentiation were trypsinized at day 2 to 5. 8 different samples were collected: entire cultures at day 2, 3, 4 and 5 (day 5 was represented in biological triplicates) and two FACS- purified T-TagBFP+/Sox1-GFP- and Sox1-GFP+ subpopulations from day 4. The solution was pelleted for 1 min at 1,000g and resuspended in D-PBS+0.04% Bovine Serum Albumin and strained through a 40 μm filter. For each sample, 8,000 to 10,000 cells of a single-cell suspension (of concentration 1,000 cells/μl) were loaded on a Chromium Controller (10x Genomics). For differentiation towards a primitive streak-like fate, mouse embryonic stem cells 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. Emulsions were performed on a Chromium Controller (10x Genomics). Libraries were prepared using the the Chromium Single Cell 3' Reagent Kit (10x Genomics) with v3 Chemistry according to the manufacturer's instructions.