GSM5208043: snRNAseq_CTRL_1; Mus musculus; RNA-Seq
All steps for tissue dissociation were performed on ice. Both hippocampi of the same animal were chopped briefly in 100 μL lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal, 0.2 U/ul recombinant RNase inhibitor in nuclease-free water) and immediately transferred to a dounce homogenizer filled with 1 mL lysis buffer. Tissue was homogenized by 15-20 strokes with each pestle A and pestle B. 2 mL of lysis buffer was added to the dounce homogenizer and the solution was incubated for 5 minutes. The dounce homogenizer was filled with 4 ml wash buffer (1% BSA. 0.2 U/ul recombinant RNase inhibitor in PBS) and homogenate was passed through a 40 µm filter before adding 8 ml wash buffer.  The sample was centrifuged at 500 g for 5 minutes at 4 °C and the pellet was resuspended in 7 mL nuclei suspension buffer (1% BSA, 0.2 U/µl RNaseInhibitor (Superasin) in PBS). Then the sample was centrifuged at 500 g for 5 minutes at 4 °C. The pellet was resuspended in 1 mL nuclei suspension buffer and filtered through a 40 µm, 30 µm and 10 µm filter. Nuclei count was assessed manually by Trypan blue staining and adjusted to a final concentration of 1000 nuclei/µL. cDNA synthesis and library preparation were conducted following the Chromium Single Cell 3' Reagent Kit v3 User Guide (CG000183 Rev B, 10X Genomics). 14 PCR cycles were performed for cDNA amplification. single-nucleus mRNA sequencing