Human skeletal muscle transcriptome (39 independent analyses). Human skeletal muscle transcriptome (39 independent analyses)

The annotation of the Affymetrix HTA 2.0 array was updated to optimise the detection of RNA in human skeletal muscle samples by removing invalid and low signal-high-variance probes Human primary skeletal muscle cells were isolated from muscle biopsies from healthy adults as previously described (Crossland et al. 2016; Crossland et al. 2017). Myogenic cell enrichment used magnetic-activated cell sorting (MACS) and anti-CD56 microbeads (130-050-401; Miltenyi Biotec). Myogenic purity was assessed through measurement of fractional desmin positivity (Crossland et al. 2016; Crossland et al. 2017). Cells were washed in PBS and fixed in ice-cold 1:1 acetone/methanol, before being blocked in 5% (v/v) goat serum for 30 min at room temperature. Cells were subsequently incubated with rabbit anti-desmin monoclonal antibody (ab32362; Abcam) for 1h at room temperature, washed, and incubated with anti-rabbit TRITC-conjugated secondary antibody as previously detailed (Crossland et al. 2016; Crossland et al. 2017). Finally, cells were washed in PBS and mounted/DAPI-stained using FluoroshieldTM mounting medium with DAPI. Myoblasts at passage 5-6 were cultured on Collagen Type I-coated 6-well dishes in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Life Technologies) containing 20% (v/v) fetal bovine serum (FBS, Sigma-Aldrich), 1% (v/v) antibiotic-antimycotic (AbAm) solution and 4mM L-glutamine (Life Technologies). Cells were maintained at 37oC with 5% CO2. Once cells reached ~95% confluency, differentiation was induced by switching the medium to DMEM/F-12 containing 2% (v/v) horse serum (Sigma-Aldrich), 4mM L-glutamine and 1% (v/v) AbAm solution (Life Technologies). Six days following the initiation of differentiation, a media change was carried out, using differentiation media supplemented with long R3 IGF-1 (10 ng/ml; Sigma-Aldrich), with or without 100 nM rapamycin (Sigma-Aldrich). DMSO (final concentration 0.01% (v/v)) was used as a vehicle control for rapamycin. Samples were collected at 0h baseline and following 4h and 24h treatment. Overall design: The samples are profiles from cultured human primary myotubes (32 independent analyses + 7 baselines (8th sample, GSM3753798 is an outlier))

Affymetrix HTA 2.0芯片的注释已完成更新，通过移除无效探针与低信号高变异探针，以优化人类骨骼肌样本中RNA的检测效能。研究人员按照此前报道的方法（Crossland等，2016；Crossland等，2017），从健康成人的肌肉活检组织中分离得到原代人骨骼肌细胞。采用磁激活细胞分选（magnetic-activated cell sorting, MACS）结合抗CD56微珠（130-050-401；Miltenyi Biotec）进行肌源性细胞富集，并通过检测结蛋白阳性率评估肌源性细胞纯度（Crossland等，2016；Crossland等，2017）。具体免疫荧光染色步骤如下：先用磷酸盐缓冲液（PBS）洗涤细胞，再用冰浴的1:1丙酮/甲醇混合液固定细胞，随后于室温下用5%（体积分数）山羊血清封闭30分钟；之后将细胞与兔抗结蛋白单克隆抗体（ab32362；Abcam）于室温下孵育1小时，洗涤后再与抗兔TRITC标记二抗孵育，详细操作参见此前文献（Crossland等，2016；Crossland等，2017）；最后用PBS洗涤细胞，使用含DAPI的Fluoroshield™封片介质进行封片及DAPI染色。取第5~6代成肌细胞，接种于Ⅰ型胶原蛋白包被的6孔培养板中，培养于含20%（体积分数）胎牛血清（fetal bovine serum, FBS，Sigma-Aldrich）、1%（体积分数）抗生素-抗真菌剂（AbAm）溶液及4mM L-谷氨酰胺（Life Technologies）的达尔伯克改良伊格尔培养基/营养混合物F-12（Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12, DMEM/F-12；Life Technologies）中，培养条件为37℃、5% CO₂。当细胞汇合度达到约95%时，更换培养基为含2%（体积分数）马血清（Sigma-Aldrich）、4mM L-谷氨酰胺及1%（体积分数）AbAm溶液（Life Technologies）的DMEM/F-12，以诱导细胞分化。分化启动6天后，更换为添加了长R3胰岛素样生长因子-1（long R3 IGF-1，10 ng/ml；Sigma-Aldrich）的分化培养基，分别设置添加100 nM雷帕霉素（Sigma-Aldrich）与不添加雷帕霉素两组；以终浓度为0.01%（体积分数）的二甲基亚砜（dimethyl sulfoxide, DMSO）作为雷帕霉素的溶剂对照。分别于0小时基线时刻、处理4小时及24小时后收集样本。整体实验设计：本数据集的样本来源于培养的原代人肌管，共包含32次独立检测分析及7份基线样本（第8份样本GSM3753798为异常值）