GSM7803235: MN_sample1_358_N12_R1; Danio rerio; RNA-Seq
Adult animals (7 weeks old) of either sex were deeply anesthetized in a slush of frozen extracellular solution containing (in mM): 134 NaCl, 2.9 KCl, 2.1 CaCl2, 1.2 MgCl2, 10 HEPES and 10 glucose, with pH of 7.8 adjusted with NaOH and osmolarity of 290 mOsm. The spinal cord was quickly dissected in the slush of frozen extracellular solution and collected. Two samples were prepared from the Tg(islet1a:GFP) line, and two samples were prepared from the Tg(chx10:GFP) line. For each sample, 6 to 10 intact isolated spinal cords were incubated in 1 ml of DMEM-F12 medium (Thermo Fisher, #11039021, osmolarity adjusted to 280-280 mOsm) containing papain 10 U/ml (Worthington-biochem, #LK003178) on a heated shaker at 37°C for 15 min. DMEM/F-12 (1 ml, 280-290 mOsm) was added to stop the enzymatic reaction. The sample was centrifuged at 300 g at 4°C for 5 min, and then re-suspended in 0.5 ml of DMEM/F-12 (280-290 mOsm) after removal of the supernatant. Following mechanical trituration using fire-polished Pasteur pipettes, the cell suspension was filtered through a cell 16 strainer (40 μm). The sample was kept at room temperature for 20 min after the addition of 0,1 ml of the nuclear DNA stain DRAQ5 (Thermo Fisher, #65-0880-92). Using fluorescence activated cell sorting (FACs), cells positive for GFP and DRAQ5 in each sample were sorted into a 384-wells plate containing a mild hypotonic lysis buffer (0.2% Triton X-100, 2 U/ml RNase inhibitor) and immediately snap-frozen on ice, then stored at -80°C. Smart-Seq2