GSM8290088
The tissues were placed in a 5 mL tube containing 2 mL of Hibernate A®/B27®/GlutaMAX™ (HEB) medium on ice to minimize degradation. The HEB medium was transferred to a 15mL conical and kept on ice for subsequent operations. Then, 2mL of chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% Nonidet™ P40 Substitute in Nuclease-Free Water) were added to the tissue, followed by douncing 10 times with loose pestle to homogenize it. After homogenization, the tissue was lysed on ice for 15 minutes, swirling 2 – 3 times during this period. The reserved chilled HEB medium was then returned to the lysed tissue solution, and the tissue was further triturated with 5–7 passes through a 1mL pipette. The lysed tissue solution was filtered through a 30 µm cell strainer (MACS SmartStrainer; Miltenyi Biotech 130-110-915) that was prewashed with 1 mL of PBS to remove debris and clumps. Filtered nuclei were centrifuged at 500 g for 5 minutes at 4°C. The supernatant was removed, and the nuclei were washed in 1mL of Nuclei Wash and Resuspension Buffer (1× PBS with 1.0% BSA and 0.2U/µl Rnase Inhibitor). The nuclei were manually counted using Trypan blue and AO-PI staining to estimate of nucleus concentration after each centrifugation and resuspended. We performed single-nucleus 3' gene expression profiling using the Chromium Next GEM Single Cell 3' Kit v3.1 (10× Genomics, 1000268) and the Chromium Next GEM Chip G Single Cell Kit (10× Genomics, 1000120). The nuclei suspension was loaded onto the Chromium single-nucleus controller (10× Genomics) to generate single-nucleus gel beads in the emulsion, according to the manufacturer's protocol. The captured nucleus was lysed, and the released RNA was barcoded through reverse transcription in individual GEMs. The nucleus-barcoded 3' gene expression libraries were sequenced on an Illumina NovaSeq6000 system by Shanghai Biochip Co., Ltd., Shanghai, China.