GSM5116941: Allantois; Mus musculus; RNA-Seq
For Embryonic and Extra-embryonic mesoderm cells samples, embryos were digested in dissociation buffer (1% 100 mM EDTA + 4% of 2,5% Trypsin in PBS), and the two pure GFP positive populations were sorted by FACS , directly in DMEM F-12 supplemented with 10% FBS and 15 mM HEPES. For ExE structures samples, embryos were pooled and incubated at 4°C in pancreatic/trypsin solution (2,5% of pancreatic enzymes + 0,5% Trypsin in PBS) for 18min then washed in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Allantois, amnion, and chorion were manually dissected in PBS supplemented with 0,04% BSA with finely sharpened forceps and tungsten needles. ExE structures were then incubated at 37°C in accutase+0,25% trypsin for 20 min (with gentle agitation at 10 min). 30% DMEM/F-12 with HEPES, 20% FBS, and 50% 4mM EDTA were added to the mix. Clumps were triturated by mouth pipetting and the solution was filtered in a non-binding Eppendorf tube with Flow MI cell 40 µm to remove debris. The solution was centrifuged at 450g for 4 min. Cells were resuspended in 50 µL in FHM. Samples were loaded on a chromium chip and processed using the Chromium Single Cell 3' Reagent kits v3 (10x Genomics) following manufacturer's recommendations. Multiplexed libraries were obtained following manufacturer's recommendations of Chromium Single Cell 3' Reagent kits v3 (10x Genomics) and loaded on a Novaseq 6000 (Illumina).