GSM8919703: human adult (1350); chimpanzee adult (Frits); Homo sapiens; RNA-Seq
50 mg of tissue was pulverized in liquid nitrogen using a pestle and mortar on cry ice. Powdered tissue was then homogenized in 2 mL EZ buffer (Nuclei isolation kit, Sigma NUC101) in a glass douncer (Kontes Glass Co.), followed by a 5' incubation on ice. Homogenates were centrifuged for 15' at 65 x g at 4 oC, resuspended in 2 mL EZ buffer and incubated for 5' on ice. Samples were centrifuged again for 15' at 65 x g at 4 oC, resuspended in 4 mL nuclear suspension buffer (NB: 0.01% BSA, 1x 'complete' protease inhibitor cocktail (Roche) in MQ) and filtered through a 40 µM cell strainer. Following this, samples were centrifuged for 15' at 65 x g at 4 oC. to separate the nuclei from other cell organelles after which a density gradient was created with OpitPrep (Sigma D1556). Pellets were resuspended in 30 mL NB after which 10 mL of 30% OptiPrep was carefully added at the bottom with a syringe and needle, followed by another 5 mL layer of 60% OptiPrep underneath. Samples were centrifuged for 15' at 1,000 x g at 4 oC. Nuclei were retrieved from between the 30% OptiPrep and PBS layers with a syringe and needle and checked for quality under a bright field microscope. Nuclei were stained using Hoechst-33342 (Molecular Probs, 1:1000 dilution) and filtered through a 35 µm strainer (Corning) before sorting. Sorting was performed using a FACS Fusion to sort for DAPI+ positive nuclei and to remove leftover debris and doublets. After sorting, nuclei were spun for 5 min at 500×g and resuspended in PBS + 1% BSA to be loaded on the 10x microfluidic chip device. To limit the influence of technical variation we combined cells from separate species on a 1:1 cell count basis per sequencing sample for processing using the 10x Chromium V3 single cell RNA Gene Expression kit according to the manufacturer's protocols. Nuclei were loaded aiming to recover 10,000 nuclei per sample. Single cell RNA-Seq samples were sequenced on an Illumina Hiseq 2500.