概要: JAK inhibitors dampen activation of interferon-stimulated transcription of ACE2 isoforms in human airway epithelial cells (RNA-seq)
项目整体设计: SARS-CoV-2 infection of human airway epithelium activates genetic programs leading to progressive hyperinflammation in COVID-19 patients. Here, we report on transcriptomes activated in primary airway cells by interferons and their suppression by Janus kinase (JAK) inhibitors. Deciphering the regulation of the angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2, is paramount for understanding the cell tropism of SARS-CoV-2 infection. ChIP-seq for activating histone marks and Pol II loading identified candidate enhancer elements controlling the ACE2 locus, including the intronic dACE2 promoter. Employing RNA-seq, we demonstrate that interferons activate expression of dACE and, to a lesser extent, the genuine ACE2 gene. Interferon-induced gene expression was mitigated by the JAK inhibitors baricitinib and ruxolitinib used therapeutically in COVID-19 patients. Through integrating RNA-seq and ChIP-seq data we provide an in-depth understanding of genetic programs activated by interferons, and our study highlights JAK inhibitors as suitable tools to suppress these in bronchial cells.
Human small airway epithelial cells (SAEC) obtained from Lifeline Technology (FC-0016) were expanded using the complete BronchiaLifeTM media kit (Lifeline Technology, LL-0023). All culture wares were pre-coated in 30 mg/ml of Fibronectin (ThermoFisher Scientific, 33016015) for at least 1h at room temperature.
处理方案:
Cytokines (10 ng/ml; Human IFNb, 300-02BC; Human IFNg, 300-02; Human IL6, 200-06; Human IL7, 200-07; Human Growth hormone, 100-40, Peprotech; Human IFNa2b, 78077.1, Stem Cell Technologies; Human IFNl3, 5259-IL-025, R&D systems) were treated in respective culture media and the cells were incubated for 12hr in 5% CO2 atmosphere at 37°C. Jak inhibitors, 10 mM of either Baricitinib (HY-15315A, MedChemExpress) or Ruxolitinib (HY-50856A, MedChemExpress), were added to BronchiLiafeTM media with or without IFNb and incubated for 12hrs.
提取方案:
Total RNA was isolated from the cytokine-stimulated cells using PureLink™ RNA Mini Kit (Invitrogen) and libraries for sequencing were prepared according to the manufacturer’s instructions with TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina, RS-122-2301).
Libraries for sequencing were prepared using standard Illumina protocols.
建库方案:
Libraries for sequencing were prepared according to the manufacturer’s instructions with TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold (Illumina, RS-122–2301)
测序信息
分子类型:
rRNA- RNA
库的片段类型:
PAIRED
库的链类型:
Forward
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 3000
链特异性:
Specific
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
Activation of Interferon-Stimulated Transcriptomes and ACE2 Isoforms in Human Airway Epithelium Is Curbed by Janus Kinase Inhibitors.
