title: Trained and untrained human skeletal muscle single-nucleus RNA-sequencing from individuals with type 2 diabetes and control
design: Trained and untrained human skeletal muscle single-nucleus RNA-sequencing from individuals with type 2 diabetes and control
library_construction_protocol: The muscle biopsies have already be analysed in a previous study (Dela et al., Acta Physiol (Oxf), 2019, doi: 10.1111/apha.13245. Epub 2019 Feb 14. PMID: 30585698). In summary, ten sedentary males with T2D and ten healthy males matched for age, body mass index (BMI), and peak oxygen uptake (V̇O2peak) performed two weeks (eight sessions) of one-legged HIIT on a bicycle ergometer. Each session included ten 1-minute intervals at >80% of maximal heart rate, interspersed with one minute of rest. Forty hours after the last exercise bout, lateral vastus muscle biopsies from the trained and untrained legs were obtained. The muscle biopsies were obtained using the Bergström technique and visible connective tissue was immediately removed. One portion of the muscle was snap snap-frozen in liquid nitrogen while an-other was embedded in OCT/Tissue-Tek precooled in liquid nitrogen. Nuclei were isolated using a modified version of the Nuclei Isolation Kit: Nuclei PURE Prep, cat. no. NUC201 from Sigma-Aldrich. The entire protocol was carried out on ice. The tissue was lysed in ice-cold lysis buffer (Nuclei Pure Lysis Buffer, Sigma cat. no. L9286, with 1 mM DL-Dithiothreitol (DTT), Sigma cat. no. D9779, and 0.01% Triton X-100, Sigma cat. no. T1565). Homogenized for 30 seconds using an ULTRA-TURRAK TP18/10 homogenizer (20,000 rpm) for 10 minutes and filtered. Nuclei were pelleted by centrifugation and washed in wash buffer (1x DPBS, no calcium, no magnesium, Gibco cat. No. 14190, with 1% Human Serum Albumin Sigma cat. No. A1887, and 0.2 u/µL RNasin Plus RNase Inhibitor [40 u/µL], Promega cat. No. 2611). The lysate was then mixed with 1.8 M Su-crose Cushion Solution (mix of Nuclei PURE 2 M Sucrose Cushion Solution, Sigma cat. no. S9308, Nuclei PURE Sucrose Cushion Solution, Sigma cat. no. S9058 and 0.83 mM DTT). The mixture was then loaded on top of 1.8 M Sucrose Cushion Solution, and centrifuged. The supernatant was re-moved and nuclei-containing pellet was resuspended, centrifuged and filtered. The final pellet was resuspended in approximately 200 µL wash buffer and counted using NucleoCounter NC-200 (ChemoMetec cat. no. 900-0201) to determine nuclei concentration. For isolation of single-nucleus RNA, the samples were run using the 10X Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 on a 10X Chromium Controller according to manufacturer's instructions (protocol version “CG000204_ChromiumNextGEMSingleCell3_v3.1_Rev_D”)