{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Hashem Mohammdian"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-14804"],"description":["This study explores how increased matrix stiffness alters fibroblast transcription and its impact on the lineage specification of fibroblast subpopulations in fibrotic skin. Fibroblasts from healthy and systemic sclerosis patients were cultured in a collagen I-based 3D system with varying matrix stiffness levels, and RNA sequencing was performed to identify stiffness-responsive genes."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Sequencing - Index-coded samples were clustered on a cBot Cluster Generation System using the PE Cluster Kit cBot-HS (Illumina) following the manufacturer's protocol. The libraries were then sequenced on an Illumina platform, generating paired-end reads.","Nucleic Acid Extraction - The collagen matrices were dissolved in RA1 lysis buffer provided with the NucleoSpin RNA isolation kit (Macherey-Nagel, Düren, Germany). RNA was isolated according to the manufacturer's protocol. RNA concentration and purity were determined using NanoDrop (Thermo Fisher Scientific).","Sample Collection - Skin biopsies were obtained from patients with systemic sclerosis according to the 2013 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) criteria. Written informed consent was obtained from all participants. The study was approved by the Ethics Committee of the University of Erlangen-Nuremberg. Fibroblasts were isolated from punch biopsies. The tissue was subjected to a 3-hour degradation process at 37 °C using Dispase II (Boehringer-Mannheim, Rotkreuz, Schweiz), followed by filtration through a 70 µm filter and centrifugation. The cell pellet was resuspended in DMEM/F12 medium, containing 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, 2.5 µg/mL amphotericin B, 25 mM HEPES, 2 mM glutamine (all Thermo Fisher Scientific, Dreieich, Germany) and 0.2 mM ascorbic acid (Sigma-Aldrich, Steinheim, Germany). The suspension was then incubated in 75 cm² cell culture flasks at 37 °C, 90% relative humidity and 5% CO₂ concentration and further cultivated.","Library Construction - Sequencing libraries were prepared using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s instructions, with index codes added to assign sequences to individual samples.","Growth Protocol - 10x DMEM (Sigma-Aldrich) and cells resuspended in 1x DMEM containing supplements (HEPES, amphotericin B, penicillin, streptomycin, L-Glutamin, FCS) (all Thermo Fisher Scientific, Dreieich, Germany) were added to a collagen type I mass (Corning Collagen I Rat Tail, concentration: 10.98 mg/mL, Corning, USA). One batch of collagen solution was used throughout the entire experiment. Supplements were added in quantities that resulted in the same final concentrations in the collagen matrices as were present in the 2D preculture. Final collagen concentration was 3.0 mg ml−1. Each matrix contained 100.000 cells. To prevent premature polymerization of the collagen, the experiment was conducted on ice at acidic pH until the intended polymerization step, which was initiated by adjusting the pH to neutral. To create collagen matrices with increasing stiffness, 300 µL of the collagen-medium-cell suspension were polymerized in coated microdishes for one hour at 16°C, 27°C, or 37°C. To ensure sample comparability one matrix of each stiffness/polymerization temperature was prepared from one batch of ready pipetted collagen-medium-cell suspension. Following polymerization, the matrices were overlaid with DMEM/F12 medium and incubated at 37 °C in 90% humidity and 5% CO₂ for 48 hours."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Quantification was performed using FeatureCounts v1.5.0-p3. Normalization was done using DESeq2 (1.42.0).","Sequence Alignment - Clean paired-end reads were aligned to the reference genome (GRCH38 p12) using HISAT2."],"omics_type":["Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["RNA-seq of coding RNA"],"species":["Homo sapiens"],"pubmed_authors":["Alina Mihaela Ramming","Andreas Ramming","Ludwig Ueberall","Hashem Mohammdian"],"additional_accession":[]},"is_claimable":false,"name":"RNA-seq of human skin fibroblasts from healthy and systemic sclerosis patients with varying matrix stiffness","description":"This study explores how increased matrix stiffness alters fibroblast transcription and its impact on the lineage specification of fibroblast subpopulations in fibrotic skin. Fibroblasts from healthy and systemic sclerosis patients were cultured in a collagen I-based 3D system with varying matrix stiffness levels, and RNA sequencing was performed to identify stiffness-responsive genes.","dates":{"release":"2026-01-01T00:00:00Z","modification":"2026-01-01T02:02:05.026Z","creation":"2025-02-03T21:11:46.15Z"},"accession":"E-MTAB-14804","cross_references":{"ENA":["ERP168909"],"EFO":["EFO_0002944","EFO_0004170","EFO_0003789","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0003738","EFO_0004184"]}}