GSM7820722: MCAO-QO-83-1, replicate1, snRNA-seq; Mus musculus; RNA-Seq
For single-nucleus RNA sequencing experiments, we dissected the nuclei from frozen tissues (mouse) with Lysis buffer (10 mM Tris-HCl (pH7.4), 10 mM NaCl, 3 mM MgCl2) plus RNase inhibitor, liberated cellular nuclei by mechanical force with a glass dounce tissue grinder. Nuclei were resuspended in a Nuclei Wash & Resuspension Buffer (1% BSA Solution, 0.2U/μL RNase Inhibitor, 1XPBS). Trypan blue and the Countess II FL Automated Cell Counter/microscopy were used to assess nucleus viability and concentration. Lastly, target nuclei concentration of 700-1,200 nuclei/µL was achieved for loading onto the chromium controller of the 10xGenomics platform. Approximately, 1.6x104 isolated nuclei and enzyme gel-beads were packed into a single oil droplet for single-cell RNA-seq library construction. Cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) at a concentration of 700-1200cells/uL (Single Cell 3′ library and Gel Bead Kit v.3) as described in the manufacturer's protocol. Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT clean-up, complementary DNA amplification and library construction were all performed as per the manufacturer's protocol.