title: GSM6721947: denervation gastrocnemius snATAC-seq  3; Mus musculus; ATAC-seq
library_construction_protocol: 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 (100~150mg) 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.  Following centrifugation, the pellet was resuspended in Nuclei Buffer (10 × Genomics, Chromium Single-Cell ATAC kit ) according to the manufacturer's protocol. 6 snATAC-seq libraries were created with 10X Genomics Chromium Single Cell ATAC v1 chemistry following nuclear dissociation according to the manufacturer's protocol