snRNA-seq of the developing human female and male gonads across prenatal development
snRNA-seq of the developing human female and male gonads across prenatal development
Frozen tissue was homogenized on ice in a buffer containing sucrose, KCl, MgCl2, Tris-HCl (pH 8), IGEPAL, DTT, Murine RNase Inhibitor, SUPERase-In, and cOmplete Protease Inhibitor Cocktail. The tissue was disrupted by trituration and/or using a micropestle. After a brief incubation, unlysed tissue debris was removed by low-speed centrifugation (100g for 1 minute at 4°C). The supernatant was then centrifuged at 400g for 4 minutes to separate the nuclei (pellet) from the supernatant. Nuclei were washed once or twice in the homogenization buffer and resuspended in a storage buffer containing sucrose, KCl, MgCl2, Tris-HCl (pH 8), Murine RNase Inhibitor, SUPERase-In, and cOmplete Protease Inhibitor Cocktail. If needed, the nuclei were filtered using 40 µm Flowmi strainers. Nuclei concentration was determined by staining with Hoechst DNA dye or propidium iodide and counting on a Countess II FL Automated Cell Counter. Following the nuclei count, approximately 15,000 nuclei from each nuclei suspension were used for generating snRNA libraries. RNA was extracted from the cytoplasm extracts or nuclei suspensions by mixing them with RLT buffer (supplemented with 40 mM DTT) and 100% ethanol in a 2:7:5 ratio. The RNA was then purified using the RNeasy Micro Kit from Qiagen. The quality of the extracted RNA was assessed using a Fragment Analyzer (Advanced Analytical), and all samples had RNA quality numbers (RQN) above 8. Single-cell barcoding and library preparation were performed using Chromium Single Cell 3' Reagent Kits (v3 chemistry) and the Chromium Controller instrument (10x Genomics), following the manufacturer's protocols. The cDNA was amplified using 12 PCR cycles. The Qubit Fluorometer (Thermo Fisher Scientific) was used to quantify the libraries, and the average fragment size was assessed using a Fragment Analyzer.