Additional file 1 of CircUBE2Q2 promotes differentiation of cattle muscle stem cells and is a potential regulatory molecule of skeletal muscle development

Additional file 1: Figure S1. Isolation, culture and differentiation of Guangxi cattle muscle stem cells (A) Cattle fetus around 3 months old. (B) Primary muscle stem cells cultured in vitro for 48 hours. (C) GM sample (proliferation) of MuSCs. (D) DM sample (differentiation) of MuSCs. (scale bars = 100/200 μm). Figure S2. The workflow of RNA-seq. Figure S3. Cluster analysis of differentially expressed RNA in MuSCs.(A–D) Volcano plots (below) displaying the differentially expressed transcripts and the hierarchical cluster analysis is shown above of each panel which displays the differential expression of RNAs in three DM samples and three GM samples of MuSCs. (A) mRNAs, (B) miRNAs, (C) lncRNAs and (D) circRNAs. The blue and yellow dots represent downregulated and upregulated RNAs in DM of MuSCs respectively, when compared with GM. The grey dots indicate no significant difference. Figure S4. Characteristics of circular RNA in MuSCs of Guangxi Cattle. (A) Distribution of genomic regions from where the detected circRNAs were derived. (B) Chromosomal distribution of circRNAs. (C) Distribution of the number of circRNAs per gene. (D) Distribution of sample expression for circRNAs. (E) Length distribution of cricRNAs. (F) SRPBM distribution of circRNAs. Figure S5-B. Cell transfection of overexpression vector pK25-circUBE2Q2 and visualization of the efficiency of circUBE2Q2. Figure S6. Vector construction of plasmids. Figure S7. Identification results of candidate miRNAs. Figure S8. The culture and adipogenic differentiation of SVFs. Figure S9. Validation of RNAs identified from RNA-seq in cattle MuSCs. Table S1. The information of differential RNAs. Table S2. a primers for RT-PCR. b primers for RT-qPCR. c primers for PCR. d primers for vector construction.

附加文件1：图S1。广西牛肌肉干细胞（muscle stem cells, MuSCs）的分离、培养与分化：(A) 约3月龄牛胎儿；(B) 体外培养48 h的原代肌肉干细胞；(C) 肌肉干细胞的GM（增殖）样本；(D) 肌肉干细胞的DM（分化）样本（标尺=100/200 μm）。
图S2。RNA测序（RNA-seq）实验流程。
图S3。肌肉干细胞差异表达RNA的聚类分析：(A–D) 各面板下方为火山图，用于展示差异表达转录本；面板上方为层级聚类分析结果，呈现3份肌肉干细胞分化组（DM）与3份增殖组（GM）的RNA差异表达情况。其中(A)为信使RNA（mRNAs）、(B)为微小RNA（miRNAs）、(C)为长链非编码RNA（lncRNAs）、(D)为环状RNA（circRNAs）。蓝、黄圆点分别代表相较于增殖组，分化组中表达下调与上调的RNA；灰色圆点表示无显著差异。
图S4。广西牛肌肉干细胞中环状RNA的特征：(A) 检测到的环状RNA的基因组来源区域分布；(B) 环状RNA的染色体分布；(C) 单个基因编码环状RNA的数量分布；(D) 环状RNA的样本表达量分布；(E) 环状RNA的长度分布；(F) 环状RNA的SRPBM值分布。
图S5-B。过表达载体pK25-circUBE2Q2的细胞转染及circUBE2Q2过表达效率的可视化验证。
图S6。质粒载体构建流程。
图S7。候选微小RNA的鉴定结果。
图S8。基质血管组分（stromal vascular fraction, SVFs）的培养与成脂分化。
图S9。牛肌肉干细胞中经RNA-seq鉴定的RNA的验证实验。
表S1。差异RNA信息表。
表S2。a：逆转录PCR（RT-PCR）引物；b：实时定量逆转录PCR（RT-qPCR）引物；c：聚合酶链式反应（PCR）引物；d：载体构建引物。