GSM5945760: St36_mpx; Pleurodeles waltl; RNA-Seq
For brain dissociation, animals were deeply anesthetized by submersion in 0.2% MS-222. The brain was perfused transcardially with ice-cold oxygenated Amphibian Ringer's solution (96 mM NaCl; 20 mM NaHCO3; 2 mM KCl; 10 mM HEPES; 11 mM glucose; 2 mM CaCl2; 0.5 mM MgCl2), and the animal decapitated. The whole brain or telencephalon was dissected out, and embedded in 4% LM Agarose in Ringer. The brain was then sliced coronally on a vibratome into 500 um sections, and cut into 500 um cubes in cold carbogenated Ringer. The tissue pieces were transferred to a 5 mL tube in 2.5 mL Dissociation Buffer (20 U/mL Papain, 200 U/mL DNase, 5 ug/mL Liberase, 1 uM TTX), and incubated at RT for 30 minutes on a rotator. Following enzymatic dissociation, the tissue was mechanically dissociated by trituration with fire polished, silanized glass pipettes of decreasing tip diameter on ice. The supernatant was passed through a 100 um cell strainer, with fresh carbogenated Hibernate A-Ca media added between each subsequent pipette until a uniform suspension was obtained. Hibernate A-Ca was added up to a total volume of 20 mL, and the solution was passed through a 70 um cell strainer. To the bottom of the tube, 5 mL of 4% BSA in Hibernate A-Ca - phenol red was added. To filter out cell debris, the cell suspension was centrifuged through the density gradient at 300xg (4°C) for 5 min. The supernatant was removed, and the pellet resuspended in 50-100 uL Hibernate A-Ca - Mg. The cell concentration was determined by counting on a Fuchs- Rosenthal chamber with trypan blue, and diluted to 1000 cells/uL in Hibernate A- Ca - Mg. Cells were multiplexed using the Chromium Next GEM Single Cell 3' v3.1: Cell Multiplexing kit The cells were loaded into a 10x Chromium Chip G with a targeted cell recovery of 5000-8000 cells for GEM Generation and cell barcoding. Single cell RNA-seq libraries were prepared with 10x Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.0 or v3.1 (Dual Index)