概要: Purpose: To better characterise the population structure of primary bovine retinal microvascular endothelial cells (RMECs) based upon their individual transcriptomes.
Methods: Individual RMECs were captured on the Fluidigm C1 system (Fluidigm), cDNA libraries were prepared using a Nextera XT kit and sequencing performed on a NextSeq (Illumina).
Results: Application of a single cell RNA-seq analysis workflow showed that RMECs form a relatively homogeneous population in culture, with the main subgroup being proliferating cells. Expression of markers from along the arteriovenous tree suggests that most cells originate from capillaries. An in silico model of the blood retinal barrier was created, including junctional proteins not previously reported within the retinal vasculature. Numerous alternative splicing events involving exons within microvascular barrier genes were observed and in many cases individual cells expressed exclusively one isoform.
Conclusions: We have optimised a workflow for single-cell transcriptomics in primary RMECs. Our results have provided fundamental insights into the genes involved in retinal microvascular barrier formation
项目整体设计: Single cell RNA-seq analysis of retinal microvascular endothelial cells from two separate Fluidigm C1 runs, 49 cells from run 1 and 51 cells from run 2.
RMECs were isolated from bovine retinas according to (Stitt, Chakravarthy, Archer, & Gardiner, 1995). RMECs were maintained in DMEM supplemented with 0.1mg/ml Primocin, 0.38渭g/ml insulin, 1mg/ml heparin and 10% (v/v) porcine serum (PS). BRECs were passaged at approximately 80% confluency in a 1:3 ratio and grown until confluency at passage 3 for Fluidigm C1 runs . RMECs were dissociated form flask using TryPLE Express dissociation reagent and single cells were captured on Fluidigm C1 Microfluidics chip.
建库方案:
-
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina NextSeq 500
链特异性:
-
样本
基本信息:
样本描述:
生物条件:
实验变量:
方案:
测序信息:
质量评估:
数据来源
GEN样本编号
GEN数据集编号
系列编号
项目编号
样本编号
样本名称
生物样本编号
样本访问号
实验访问号
释放时间
提交时间
最后更新时间
物种
种族
族裔
年龄
年龄单位
性别
来源名称
组织
细胞类型
细胞亚型
细胞系
疾病
疾病状态
发育阶段
突变/变异
表型
Condition Detail
生长方案
处理方案
提取方案
建库方案
分子类型
库的片段类型
链特异性
库的链类型
加标（Spike-In）
测序方法
测序平台
测序仪型号
细胞数
测序片段数
碱基数
平均测序片段长度_1
平均测序片段长度_2
唯一比对率
多重比对率
覆盖度
文章
Single-cell transcriptomic profiling provides insights into retinal endothelial barrier properties.
