Gene Expression
Nebulas
Gene Expression NebulasSummary: 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.
Overall Design: 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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| Growth Protocol: | 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. |
| Treatment Protocol: | - |
| Extract Protocol: | Total RNA was purified with the RNeasy Mini Kit (Qiagen), according to the manufacturer's instructions. |
| Library Construction Protocol: | 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. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 2000 |
| Strand-Specific: | Unspecific |