title: GSM8392728: GP muscle from KIC mice [scRNA]; Mus musculus; RNA-Seq
library_name: GSM8392728
library_construction_protocol: Skeletal muscles were dissected, snap-frozen, and stored in liquid nitrogen until the single nucleus multi-ome procedure was performed. Three control and three KIC GP muscles were pooled together. Muscles were thawed for 5 min on ice and minced with scissors in 1 mL cold lysis buffer (10 mM Tris- HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NonidetTM P40, 1 mM DTT, 1 U/μl RNase inhibitor in Nuclease-Free Water). Cold lysis buffer was then added, and muscles were lysed for 5 min at 4 °C. Cold wash buffer (PBS, BSA 2%, and 0.2 U/μl RNase inhibitor from Roche) was added, and the lysate was dounced with a loose dounce homogenizer for 20 strokes. The homogenate was then filtered with 70 μm and 40 μm cell strainers. Nuclei were pelleted by centrifugation for 5 min at 500 x g at 4 °C. Nuclei were then resuspended in wash buffer and 2 μL of Nuclear Pore Complex antibody (Biolegend, 682203) to stain all nuclei. After 15 min, nuclei were pelleted by centrifugation for 5 min at 500 x g at 4 °C. Nuclei were then resuspended in wash buffer and filtered with 30 μm cell strainers.  Nuclei were FACS sorted to exclude debris with a BD FACSAria III and the BD FACSDIVA and Flowjo v10.8.1 software. Nuclear Pore Complex-positive nuclei were collected for each condition and pelleted by centrifugation for 10 min at 500 x g at 4 °C. Nuclei were then resuspended in ATAC lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% NonidetTM P40, 0.001% Digitonin, 1% BSA, 1 mM DTT, 1U/μl RNase inhibitor in Nuclease- Free Water). After 2 min, ATAC wash buffer (10 mM Tris- HCl pH7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1 mM DTT, 1% BSA, 1U/μl RNase inhibitor in Nuclease-Free Water) was added. Nuclei were then pelleted by centrifugation for 10 min at 500 x g at 4 °C and resuspended in diluted nuclei buffer from 10x Genomics.  Nuclei concentrations were quantified with an automatic hemocytometer, and were adjusted to 2000 nuclei/μL with wash buffer. We loaded ~ 5000 nuclei per condition into the 10x Chromium Chip. We used the Next GEM single-cell Multiome ATAC+ Gene expression kit according to manufacturer's protocol. SnRNA-seq libraries were constructed by performing the following steps: cDNA Amplications, SPRIselect cleanup, Fragmentation, End repair and A tailing, SPRIselect cleanup, and Adaptor ligation, SPRIselect cleanup, Sample index PCR and SPRIselect cleanup size selection. Fragment size estimation was assessed with Agilent TapeStation D1000 (Agilent) and quantified using the QubitTM dsDNA High-Sensitivity HS assay (ThermoFisher Scientific).