Gene Expression
Nebulas
Gene Expression Nebulas概要: Systemic sclerosis (SSc) is a chronic autoimmune disease that mainly affects the connective tissue. Monocytes have been shown to be an important cell type involved in the pathogenesis of SSc. By performing RNA-sequencing analysis on whole RNA isolated from peripheral blood CD14+ monocytes obtained from SSc patients, together with healthy controls matched for sex and age, obtained from the University Medical Center Utrecht (definite SSc cohort), and the University of Milan (non-fibrotic SSc cohort), we aimed to characterize the transcriptomic landscape of monocytes of patients with (pre-clinical) systemic sclerosis. Moreover, ChIPseq data was available for a part of the subjects included in the RNA-seq analysis and the correlation between the histone marks and gene expression was studied. The samples used in this study are part of the SYSCLASS cohort.
项目整体设计: Transcriptomic profiling was performed for two SSc cohorts. For the definite SSc cohort (Utrecht) total RNA from 9 heathy controls and 25 SSc patients were subjected to RNA-seq analysis. For the non-fibrotic SSc cohort (Milan) total RNA from 9 heathy controls and 30 pre-clinical SSc patients were subjected to RNA-seq analysis.
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| 生长方案: | Monocytes (3 × 106 cells/ml) and PMNs (5 × 106 cells/ml) were cultured in RPMI 1640 (Gibco) supplemented with 10% FCS (<0.5 EU/ml; Sigma-Aldrich) and 2 mM Glu in the presence or absence of 100 ng/ml ultra-pure lipopolysaccharide (LPS, from E. coli strain O111:B4, InvivoGen, San Diego, CA, USA), 5 μM R848 (Invivogen), 1,000 U/ml IFNα CRI003B, Cell Sciences), 100 ng/ml palmitoyl-3-cysteine-serine-lysine-4 (Pam3CSK4, Invivogen), 50 μg/ml polynosinic:polycytidylic acids [poly(I:C), Invivogen], as indicated. In selected experiments, CD14+ monocytes were incubated for 30 min with 5 μg/ml Brefeldin A (BFA, Sigma-Aldrich) or 5 μg/ml αIFNAR (PBL InterferonSource, Piscataway, NJ, USA) or its isotype control antibody (mouse IgG2a), before cell stimulation. |
| 处理方案: | - |
| 提取方案: | Total RNA was purified with the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. |
| 建库方案: | RNA sequencing libraries were generated from total RNA extracted from CD14+ monocytes of SSc patients and matched HC enrolled in the “definite SSc” and “non-fibrotic SSc” cohorts, or from RNA pools of three different donors of freshly isolated and LPS-treated monocytes. |
| 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 HiSeq 2000 |
| 链特异性: | Unspecific |
| 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 |
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