title: GSM8434190: Brain and spinal cord, Stripe adult corn snake, replicate 1; Pantherophis guttatus; RNA-Seq
library_construction_protocol: A Stripe corn snake was frozen at -20°C after euthanasia. It was then thawed for the dissection of the brain and the spinal cord. The tissues were then flash frozen. Nuclei were isolated using the Nuclei EZ Prep Kit (NUC-101, Sigma). Briefly, frozen tissue was resuspended in 2mL of cold isolation buffer, dounce homogenized with a KIMBLE Dounce tissue grinder (D8938, Sigma) and incubated at 4°C during 5 minutes in the same buffer. The samples were centrifuged, resuspended and incubated in fresh buffer for 5 extra minutes. Nuclei were washed twice using 1% BSA (bovine albumin serum) PBS (phosphate-buffered saline) containing 50 U/ml of SUPERase-In (AM2696, Thermo Fisher) and 50 U/ml of RNasin (N2611Promega). Nuclei were resuspended in the appropriate volume of specific cell multiplexing oligos (CMO – CG000388, 10x Genomics,) and incubated for 5 minutes at room temperature. After adding 1.9mL of 1% BSA-PBS, nuclei were centrifuged for 10 minutes at 4°C and 500g. A second washing step was performed in order to remove the excess of CMOs. Nuclei were finally filtered through a 30μm strainer and stained with Hoechst (Invitrogen H3570, 1:500) for 5 minutes. To remove debris and only retain good quality nuclei, we used fluorescence-activated nuclei sorting (FANS) on a Beckman Coulter MoFloAstrios. CMO-labelled and sorted samples were pooled after sorting. We used approximatively 10,000 nuclei per sample and 42 μl of nuclei suspension was used for the 10x Genomics snRNA-seq preparation according to the manufacturer's instructions (10X Genomics Chromium 3′ GeneExpression Kit). The cDNA libraries were quality controlled using a 2100 Bioanalyzer and TapeStation from Agilent and sequenced using a HiSeq 2500 sequencer from the iGE3 platform at the University of Geneva.