Summary: Integration of differential expression (DE) and expression QTL (eQTL) analysis in monocyte-derived macrophages (MDMs) from systemic sclerosis (SSc) patients and healthy controls reveals (i) changes in macrophage transcriptome as an important contributor in SSc and (ii) cis-regulation in GSDMA as a disease risk in macrophages, but not skin.
Overall Design: We carried out RNA-sequencing and genome-wide genotyping in MDMs from 57 SSc patients and 15 controls. Our differential expression and expression QTL (eQTL) analysis in SSc was further integrated with epigenetic, expression and eQTL data from skin, monocytes, neutrophils and lymphocytes.
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Growth Protocol: | Human MDMs were differentiated from total blood from SSc patients and healthy donors using gradient separation (Histopaque 1077, Sigma) and adhesion purification. Following Histopaque separation, peripheral blood mononuclear cells (PBMCs) were re-suspended in RPMI (Life Technologies) and monocytes were purified by adherence for 1 h at 37 °C, 5% CO2. The monolayer was washed 3 times with HBSS to remove non-adherent cells and monocytes were matured for 5 days in RPMI containing 100 ng ml-1 M-CSF (PeproTech, UK) and 10% Fetal Calf Serum (FCS, Labtech International). Macrophage purity was confirmed by immunohistochemical assessment of CD68 and >99% cells were CD68+. |
Treatment Protocol: | - |
Extract Protocol: | Total RNA was extracted from hMDMs using Trizol (Invitrogen) and RNeasy mini kit (Qiagen) according to manufacturer's instructions, with an additional purification step by on-column DNase treatment using the RNase-free DNase Kit (Qiagen) to ensure elimination of any genomic DNA. The integrity and quantity of total RNA were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) and Agilent 2100 Bioanalyzer (Agilent Technologies). |
Library Construction Protocol: | In total, 500 ng of total RNA was used to generate RNA-seq libraries using TruSeq RNA sample preparation kit (Illumina) according to the manufacturer's instructions. Briefly, RNA was purified and fragmented using poly-T oligo-attached magnetic beads using two rounds of purification followed by the first and second cDNA strand synthesis. Next, cDNA 3' ends were adenylated and adapters ligated followed by 15 cycles of library amplification. Finally, the libraries were size selected using AMPure XP Beads (Beckman Coulter) purified and their quality was checked using Agilent 2100 Bioanalyzer. Samples were randomized to avoid batch effects and multiplexed libraries were run on a single lane (6 samples/lane) of the HiSeq 2500 platform (Illumina) to generate 100bp paired-end reads. An average of 64M reads coverage per samples was achieved. |
Molecule Type: | poly(A)+ RNA |
Library Source: | |
Library Layout: | PAIRED |
Library Strand: | Forward |
Platform: | ILLUMINA |
Instrument Model: | Illumina HiSeq 2500 |
Strand-Specific: | Specific |
Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
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