title: GSM7060357: Sheep 3 + 4, scRNAseq; Ovis aries; RNA-Seq
library_construction_protocol: cells: Dissociation of neural tissue for scRNA-seq was performed using a modified protocol from 10X Genomics (10X Demonstrated Protocol, CG00055). In brief, 2 mL of 2 mg/mL Papain (Sigma) was added to 30 mg of tissue and incubated at 37 ºC for 20 minutes. The papain solution was then removed without disrupting the tissue, leaving enough solution to cover the tissue. The tissue was dissociated using a Pasteur pipette with 2 mL of Neurobasal media (ThermoFisher Scientific), leaving cell debris to settle on the bottom. The supernatant containing the cells were transferred to a new 15 mL centrifuge tube and centrifuged at 200 xg for 2 minutes. The supernatant was removed, and the cell pellet resuspended in 1 mL Neurobasal media. 5 µL of resuspended cells was stained with 5 µL trypan blue to assess the cellular membrane intactness and number of cells was assessed using the Countess II FL Automated Cell Counter (ThermoFisher Scientific). The cell suspension was diluted by an appropriate volume of Neurobasal media to a final titer of 1000 cells/µL. Cell suspensions from the tissues isolated from animals 1+2 and animals 2+3 were pooled resulting in a total of two cell suspensions. Nuclei: Nuclei were extracted from frozen ovine brain tissue utilising an adapted protocol from Krishnaswami et al., 2016. Briefly, approximately 50-100 mg of brain tissue was transferred to a dounce homogenizer containing 1mL homogenization buffer, HB (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 1µM DTT, 1X protease inhibitor, 0.4 U/µL RNaseIn (ThermoFisher Scientific), 0.2 U/µL Superasein (ThermoFisher Scientific), 0.1 % Triton X-100). Tissue was homogenised using 5 strokes with the loose dounce pestle A, followed by 10-15 strokes of tight dounce pestle B. The homogenate was filtered through a 40 µm strainer into 5 mL Eppendorf tubes and centrifuged at 1000 xg (4 oC) for 8 minutes. The supernatant was removed and the pellet was resuspended in 250 µL of HB. A 50% - 29% iodixanol gradient (OptiPrep™ Density Gradient Medium, Sigma) was prepared to allow removal of the myelin layer. 250 µL of 50 % iodixanol was added to the nuclei-HB mixture and slowly layered on top of 500 µL of 29 % iodixanol in a new Eppendorf tube. The resultant gradient was centrifuged at 13,000 xg (4 C) for 40 minutes. The supernatant and myelin was removed and the purified nuclei pellet was resuspended in 1 mL PBS, 1 % BSA, 0.2 U/µl RNAse inhibitor. 5 µL of resuspended nuclei was stained with 5 µL trypan blue and the quality and number of nuclei was assessed using the Countess II FL Automated Cell Counter (ThermoFisher Scientific). Nuclei suspensions from animals 1+2 and animals 3+4 were pooled resulting in a total of two nuclei suspensions. Single cell and single nuclei RNA libraries were prepared using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (10x Genomics) as per manufacturer's instructions.