RNA sequence of mRNA in HUVEC cells after depleting EGFL6. RNA sequence of mRNA in HUVEC cells after depleting EGFL6

Total RNA was isolated from each thymic sample using the standard TRIzol protocol (Invitrogen, Carlsbad, CA, USA). RNA quality was examined by gel electrophoresis and with a Nanodrop spectrophotometer (Thermo, Waltham, MA, USA). For RNA sequencing, RNA samples from 9 biological replicates were separated into three independent pools, each comprised of three distinct samples at equal amounts. Strand-specific libraries were constructed using the TruSeq RNA sample preparation kit (Illumina, San Diego, CA, USA), and sequencing was carried out using the Illumina HiSeq X Ten instrument by the commercial service of Genergy Biotechnology Co. Ltd. (Shanghai, China). The raw data was delieved into NCBI’s Gene Expression Omnibius, and was handled by Perl and data quality was checked by FastQC v0.11.2. Clean reads were aligned to the chicken genome (release: Gallus gallus 4.0) from NCBI using Bowtie, with one mismatch allowed. As shown by previous study[2], the expression of the transcript was calculated by FPKM using Perl. Differentially expression transcripts (DETs) were determined using the MA-plot-based method with Random Sampling (MARS) model in the DEGseq package between different time points. The thresholds for determining DETs are P < 0.05 and absolute fold change ≥ 2. Then DETs were chosen for function and signaling pathway enrichment analysis using GO and KEGG database. Overall design: 3 samples of control group and 3 samples of knock-down group

采用标准TRIzol试剂操作流程（Invitrogen，美国加利福尼亚州卡尔斯巴德市），从每份胸腺样品中提取总RNA。通过琼脂糖凝胶电泳与Nanodrop分光光度计（赛默飞世尔科技，美国马萨诸塞州沃尔瑟姆市）检测RNA质量。针对RNA测序实验，将9份生物学重复的RNA样品均分为3个独立混合池，每个混合池由3份等量的不同样品组成。使用TruSeq RNA样本制备试剂盒（Illumina，美国加利福尼亚州圣迭戈市）构建链特异性文库，并委托中国上海劲捷生物科技有限公司（Genergy Biotechnology Co. Ltd.）采用Illumina HiSeq X Ten测序平台完成测序。原始测序数据提交至美国国家生物技术信息中心（NCBI）的基因表达综合数据库（Gene Expression Omnibus），使用Perl语言进行数据处理，并通过FastQC v0.11.2工具完成数据质量评估。将过滤得到的洁净序列（clean reads）与NCBI公布的鸡基因组（版本：Gallus gallus 4.0）进行比对，允许1个碱基错配。如先前研究[2]所示，借助Perl语言以FPKM（每百万比对片段中来自某转录本每千碱基的片段数）值计算转录本的表达水平。采用DEGseq软件包中基于随机抽样的MA图法（MARS模型），在不同时间点之间筛选差异表达转录本（DETs），筛选阈值为P值小于0.05且绝对折叠变化≥2。随后选取DETs进行GO（基因本体）与KEGG（京都基因与基因组百科全书）数据库的功能及信号通路富集分析。实验整体设计：对照组与敲低组各设置3份样品。