snATAC-seq of the developing human female and male gonads across prenatal development
snATAC-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 were used for snATAC 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. Nuclei were processed using Chromium Single Cell ATAC Reagent kits (v1) and the Chromium Controller instrument (10x Genomics; RRID:SCR_019326), following the manufacturer's guidelines. The process included tagmentation, single cell barcoding, and library preparation. The libraries were then amplified through 10 PCR cycles and quantified using a Qubit Fluorometer (Thermo Fisher Scientific; RRID:SCR_018095). The average fragment size of the libraries was determined using a Fragment Analyzer (Agilent; RRID:SCR_019417).