Comparative transcriptomic analyses provide insights into key genes involved in niche-associated functions of primary murine LEC and BEC in homeostasis

Purpose: The goal of this study is to compare the transcriptome profiles of freshly isolated primary murine endothelial populations, namely the lymph node-derived lymphatic endothelial cell (LN-LEC) and blood endothelial cell (LN-BEC), and the diaphragm-derived LEC (D-LEC) in homeostasis. Methods: LN-LEC and LN-BEC total RNA samples were prepared by pooling inguinal, axillary, brachial, cervical, and mesenteric LNs from wild-type C57BL/6 (B6) mice (6-8 weeks of age, purchased from NCI), followed by mechanical and enzymatic digestion, CD45- cell lineage enrichment using MACS beads (deplete S protocol in AUTOMACS), and sorted by flow cytometry based on CD45, CD31, and podoplanin expressions using Influx (BD) into RNA Protect (Qiagen). D-LEC total RNA samples were prepared by mechanical and enzymatic digestion of tissues, followed directly by flow cytometry sorting as described above. Total RNA extraction was performed using RNAeasy mini kit (Qiagen) as per manufacturer's instructions. The cDNA library preparation and sequencing were performed by the Genomic Services Laboratory at Hudson Alpha, USA. Briefly, purified total RNAs (RIN score of 7.0 or higher) were prepared for sequencing using the Ovation RNA-seq System V2 kit (Nugen) followed by RNA-sequencing of 100 paired-end reads using the Illumina HiSeq 2500 v4 platform. Raw RNA-sequencing read quality was assessed using FastQC and low quality regions were trimmed using Fastx-trimmer. Cleaned reads were aligned to the mouse reference genome (build mm9) using STAR and read counts on known mouse genes were calculated using featureCounts, part of the Subread package. Next, uniquely aligned reads were analyzed using the DEseq2 package in the R statistical computing environment to obtain normalized counts, estimate dispersion, and determine a negative binomial model for each gene. Differentially expressed genes (DEG) were determined using DESeq2 and the Benjamini-Hochberg False Discovery Rate procedure was used to re-estimate the adjusted p-values. Differentially expressed genes (DEGs) were subsequently identified as those with an FPKM of 1 or greater, p-adjusted < 0.05, and additionally 5X-DEG subsets were identified as those with fold-change of 5 or greater. Results: Post-sort analyses of LN-LEC, LN-BEC, and D-LEC replicates showed 92.6-98.6% purity. RNAseq yielded 48-98 million reads per replicate, with an average length of 180 nucleotides, and an average of 85.7% uniquely mapped reads. These reads mapped a total of 23284 genes, of which 15331 were considered expressed based on an average FPKM of 1 or greater in at least one cell type. Of this number, 14718, 14893, and 14384 genes were considered expressed in LN-LEC, LN-BEC, and D-LEC, respectively. Principal component analysis revealed that the transcriptional profiles of sample replicates clustered tightly, and that those of LN-LEC, LN-BEC, and D-LEC differed from each other. Conclusions: Our study provides insights into key genes involved in niche-associated functions of primary murine LEC and BEC in homeostasis. The RNA-seq data reported here may provide a conceptual framework for future comparative investigations of LN-LEC, LN-BEC, and D-LEC phenotypic expression profiles, heterogeneity, and niche-specific functions in homeostasis as well as disease. Overall design: mRNA profiles of 4 LN-LEC, 3 LN-BEC, and 3 D-LEC samples from wild-type C57BL/6 (B6) mice (6-8 weeks of age) were generated by RNA-sequencing using the Illumina HiSeq 2500 v4 platform.

### 研究目的
本研究旨在比较稳态条件下新鲜分离的原代小鼠内皮细胞群体的转录组（transcriptome）谱，具体包括淋巴结来源的淋巴内皮细胞（LN-LEC）、淋巴结来源的血液内皮细胞（LN-BEC）以及膈肌来源的淋巴内皮细胞（D-LEC）。

### 研究方法
LN-LEC与LN-BEC的总RNA样本制备流程如下：从野生型C57BL/6（B6）小鼠（6-8周龄，购自美国国家癌症研究所NCI）的腹股沟、腋下、臂部、颈部及肠系膜淋巴结中收集组织并混合，经机械与酶解法消化后，使用MACS磁珠（MACS beads，采用AUTOMACS中的S型去除方案）富集CD45阴性细胞谱系，随后通过BD Influx流式细胞仪（flow cytometry）基于CD45、CD31及podoplanin的表达特征进行分选，将目标细胞收集至Qiagen的RNA Protect试剂中。
D-LEC的总RNA样本制备流程为：对膈肌组织进行机械与酶解法消化后，直接按照上述流程完成流式细胞分选。
总RNA提取采用Qiagen的RNeasy Mini试剂盒（RNAeasy mini kit），严格遵循制造商说明书操作。
cDNA文库制备与测序由美国Hudson Alpha的基因组服务实验室完成。具体步骤为：先筛选RIN值≥7.0的纯化总RNA，使用Nugen的Ovation RNA-seq System V2试剂盒制备测序文库，随后采用Illumina HiSeq 2500 v4平台进行100 bp双端RNA测序。
使用FastQC评估原始RNA测序读段的质量，通过Fastx-trimmer修剪低质量区域。将清理后的读段比对至小鼠参考基因组（build mm9），使用Subread套件（Subread package）中的featureCounts工具计算已知小鼠基因的读段计数。
在R统计计算环境中，使用DESeq2包对唯一比对的读段进行分析，以获取标准化计数、估计离散度，并为每个基因构建负二项模型。采用DESeq2确定差异表达基因（differentially expressed gene, DEG），并通过Benjamini-Hochberg错误发现率程序校正P值。最终差异表达基因的筛选标准为FPKM值≥1、校正后P值<0.05；此外，将倍数变化≥5的DEG子集定义为5X-DEG。

### 研究结果
LN-LEC、LN-BEC及D-LEC的分选后纯度分析显示，各样本重复的纯度为92.6%-98.6%。
RNA测序每个重复样本获得4800万至9800万条读段，平均读长180 nt，平均唯一比对率为85.7%。
上述读段共比对到23284个基因，其中15331个基因在至少一种细胞类型中的平均FPKM值≥1，被判定为表达基因。其中，LN-LEC、LN-BEC及D-LEC中分别包含14718、14893及14384个表达基因。
主成分分析结果表明，样本重复的转录组谱紧密聚类，且LN-LEC、LN-BEC与D-LEC的转录谱彼此存在显著差异。

### 研究结论
本研究揭示了稳态条件下原代小鼠淋巴内皮细胞与血液内皮细胞的微环境相关功能所涉及的关键基因。本文报道的RNA测序数据可为未来针对LN-LEC、LN-BEC及D-LEC在稳态及疾病状态下的表型表达谱、异质性及微环境特异性功能的比较研究提供概念框架。

### 整体实验设计
本研究对来自6-8周龄野生型C57BL/6（B6）小鼠的4个LN-LEC样本、3个LN-BEC样本及3个D-LEC样本进行mRNA转录组分析，采用Illumina HiSeq 2500 v4平台完成RNA测序。