title: GSM9383558: Mixed cells from medial preoptic area, from control sexually-naïve male, biol rep 1; Rhabdomys pumilio; RNA-Seq
library_construction_protocol: Nuclei were isolated from frozen tissue punches in batches of 4 samples, counterbalanced by phenotype. The protocol for nuclei preparation was modified from 10X protocol GC000393. For more details, refer to Supplementary Methods. Samples were removed from storage (-80ºC) and immediately submerged in 500µL of ice-cold homogenization buffer containing RNasin Plus RNase Inhibitor (Promega N2611) and Complete, EDTA-free Protease Inhibitor Cocktail (Sigma, Roche 11873580001). Samples were Dounce homogenized 10 times in the homogenization buffer with a 2ml Kimble Kontes Dounce tissue grinder on ice. 5% IGEPAL CA-630 was added to the Dounce and each sample was further homogenized 5 times before filtering through a 40µm cell strainer (BD Falcon 352340). The filtrate was then diluted 1:1 with a 50% Iodixanol solution (OptiPrep Density Gradient Medium (Sigma D1556) diluted to 50%) and added to a 1.5mL microcentrifuge tube containing 100µl layers (from bottom to top) of 40% and 30% Iodixanol. Samples were then centrifuged at 4500rcf in a swinging bucket centrifuge at 4ºC for 45-minutes with a moderated ramp-up speed and the lowest break setting. Nuclei were then extracted in a 60µL volume from the resulting interstitial layer between the 40% and 30% Iodixanol layers. Nuclei concentrations were then determined via hemocytometer. Isolated nuclei were transferred to the Genomics Core Facility in the Lewis-Sigler Institute for Integrative Genomics at Princeton for library preparation with the 10X Genomics Chromium system and sequencing. Libraries were prepared based on an estimated input of 20,000 and recovery of ~12,535 nuclei per sample. All subsequent sequencing was carried out on an Illumina NovaSeq 6000 and samples were sequenced to a mean depth of 42,426 reads per nucleus (SD=8894) and a mean saturation of 71.64 (SD=10.01).