GSM6475260: WT_hindlimb_skeletal_muscle_E14.5 [snRNA-seq]; Mus musculus; RNA-Seq
Hindlimb skeletal muscles (without skin and bone) were dissected, snap-frozen, and stored in liquid nitrogen.  For the E14.5 embryo, 56 hindlimbs were pooled together. For the E18.5, 14 hindlimbs were pooled together. For the P5, 8 hindlimbs were pooled together. For the adult, 4 hindlimbs were pooled together. For E18.5 Cacna1s-/-, 26 hindlimbs were pooled together. Skeletal muscles were thawed for 5 min on ice. Muscles were 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, 1mM DTT, 1U/μl RNase inhibitor in Nuclease-Free Water). After 2 min, 4 ml of cold lysis buffer were added, and muscles were lysed for 5 min at +4 °C. 10 ml of cold wash buffer (PBS, BSA 2%, and 0.2 U/μl RNase inhibitor from Roche) was added, and the lysate was dounced with 20 strokes of loose pestle. The homogenate was filtered with 70 μm and 40 μm cell strainers. Nuclei were pelleted by centrifugation for 5 min at 500 × g at +4 °C. Nuclei were resuspended in 0.5ml of wash buffer and 2μl of PCM1 antibody (Sigma-Aldrich HPA023374) to stain the myonuclei. After 15 min, nuclei were pelleted by centrifugation for 5 min at 500 × g at +4 °C. Nuclei were resuspended in 0.5ml of wash buffer and 2μl of PCNA antibody (Biolegend 682203) to stain all the nuclei and 1μl of secondary antibody red Goat anti-rabbit against PCM1. After 15 min nuclei were pelleted by centrifugation for 5 min at 500 × g at +4 °C. Then the nuclei were resuspended in 300μl of wash buffer and filtered with 30μm cell strainers. Library was performed according to the manufacter's instructions (Next GEM single-cell Multiome ATAC+ Gene expression kit). Briefly, Post GEM-incubation Cleanup using DynaBeads MyOne Silane Beads was followed by Pre-Amplification PCR (72 °C for 5 min, 98 °C for 3 min, cycled 7 × 98 °C for 20 s, 63 °C for 30 s, 72 °C for 1 min). After cleanup with SPRIselect Reagent Kit, ATAC library was generated (98 °C for 45 s, cycled 8 × 98 °C for 20 s, 67 °C for 30 s, 72 °C for 20 s) and purified with SPRIselect Reagent Kit. The fragment size estimation of the resulting ATAC libraries was assessed with Agilent TapeStation D1000 HS (Agilent) and quantified using the QubitTM double stranded DNA (dsDNA) High-Sensitivity HS assay (ThermoFisher Scientific). SnATAC-seq libraries were then sequenced by pair with a HighOutput flowcel using an Illumina Nextseq 500 with the following mode: Read 1N - 63 cycles, i7 index – 8 cycles, i5 index - 24 cycles *(8 dark cycles and 16 cycles on i5), Read 2N - 63 cycles. SnRNA-seq libraries were constructed by performing the following steps: cDNA Amplification, SPRIselect cleanup, Fragmentation, End reair and A tailing, SPRIselect cleanup, and Adaptor ligation, SPRIselect cleanup, Samplex index PCR and SPRIselect cleanup size selection. The fragment size estimation of the resulting libraries was assessed with Agilent TapeStation D1000 (Agilent) and quantified using the QubitTM double stranded DNA (dsDNA) High-Sensitivity HS assay (ThermoFisher Scientific).