GSM5571587: denervation gastrocnemius snRNA-seq  1; Mus musculus; RNA-Seq
We first prepared nuclei isolation media1 (NIM1: 320 mM sucrose, 25mM KCl, 5 mM MgCl2, and 10 mM Tris buffer, pH=8.0) and homogenization buffer [NIM1 buffer containing 1 µM  DTT, 0.4 U/μL RNase Inhibitor (NEB Inc, Ipswich, MA), 0.20U/μL SUPERase-In RNase Inhibitor (Thermo Fisher scientific Inc, Waltham, MA), and 0.1% Triton X-100]. Then, Gas muscles (100~150  mg) were minced on ice with a razor blade. Muscles were resuspended in 5 mL ice-cold homogenization buffer and homogenized with a dounce grinder for 12 strokes gently on ice. The  homogenate was sequentially filtered through 70 mm and 40 mm cell strainers (pluriSelect, El Cajon,CA) and centrifuged (x1000g) for 5 minutes at 4°C to pellet the nuclei. The pellet was resuspended  with 1 mL of ice-cold wash buffer (PBS containing 2% bovine serum albumin and 0.2U/μL ribonuclease inhibitor) and then filtered through the 20μm cell strainer before centrifugation (x500g) for 5 minutes at 4°C. The collected nuclei were resuspended in 100 μL of cold wash buffer and the purity of nuclei suspensions was confirmed by microscopy with DAPI staining. RNA libraries were prepared with Chromium Single Cell 3' Reagent Kits v3 according to the manufacturer's protocol