3dpa_p
For single cell RNAseq (scRNAseq), ten spinal cords at 0 and 3dpa were isolated and transferred in Modified Ringer's (MR, 100mM NaCl, 1.8mM KCl, 2mM CaCl2, 1mM MgCl2, 5mM Hepes pH7) with 20µM Actinomycin D for 15 min to prevent de novo synthesis of mRNA [5]. The spinal cords were transferred in CMF-MR (100mM NaCl, 1.8mM KCl, 1mM EDTA and 5mM Hepes pH7), cut in small pieces using a fine scapel and incubated in 200µL of 0.5% Trypsin-EDTA without dye (GIBCO) for 30 min at 28ºC with shaking at 600rpm. After a 5 min spin at 300rcf at 4ºC, the spinal cords were incubated for 15 min at room temperature (RT) in 180µL of MR with 0.3U.µL-1 of DNaseI (D5025, Sigma). After trituration, 20µL of Collagenase IV at 50U.µL-1 was added and the samples were incubated 30 min at 28ºC with shaking at 600rpm. The samples were passed through a flame elongated capillary, applied to a 5µm strainer and collected in MR with 0.0375% BSA. The samples were pelleted by centrifugation at 300rcf at 4ºC for 5 min, washed with MR-BSA and resuspended in 35µL of MR-BSA. The cell number (4.105 to 5.105 cells.mL-1) and viability (above 90%) were estimated on a Countess Cell Counter (Invitrogen). The cells were then loaded onto the 10X Genomics platform for processing. Xenopus tropicalis tadpoles at stage NF50 were anaesthetised in 0.1% MS222 in 0.01X Marc's Modified Ringer (MMR) solution (10mM NaCl, 0.2mM KCl, 0.1mM MgSO4, 0.2mM CaCl2, 0.5mM HEPES, pH 7.4) Used 10X sample collection protocol, detailed in CG000053_CellPrepGuide_RevC.pdf Used 10X sample collection protocol, detailed in CG000053_CellPrepGuide_RevC.pdf