GSM6340269: N17, scRNA-Seq; Rattus norvegicus; RNA-Seq
The collected tissues were rinsed using 1× phosphate-buffered saline, then quick-frozen, and stored in liquid nitrogen. Nuclei of each tissue were separated by mechanical extraction. Briefly, the tissues were first thawed, infiltrated by 1× homogenization buffer (containing 30 mM CaCl2, 18 mM Mg(Ac)2, 60 mM Tris-HCl pH 7.8, 320 mM sucrose, 0.1% NP-40, 0.1 mM EDTA, and 0.2 U/µl RNase inhibitor), and cut into smaller pieces, with the single nucleus then isolated by 2 ml of Dounce homogenizer. After filtration with a 30 µm strainer, the nuclear extraction was resuspended in 1% bovine serum albumin (BSA) containing 0.2 U/µl RNase inhibitor and centrifuged at 500 × g for 10 min at 4 °C to discard cellular impurities within the supernatant. This step was repeated twice, after which the nuclei were recollected with 0.1% BSA containing 0.2 U/µl RNase inhibitor. Subsequently, 4′,6-diamidino-2-phenylindole was used to stain the nuclei and nucleus density was calculated under a fluorescence microscope for subsequent library construction. Extracted nuclei from two rat penis were mixed and subjected to single nucleus library construction using chromium single cell 3' reagent kits (v3 Chemistry) following the manufacture's protocol.