title: RNA-seq of human skin fibroblasts from healthy and systemic sclerosis patients with varying matrix stiffness
design: RNA-seq of human skin fibroblasts from healthy and systemic sclerosis patients with varying matrix stiffness
library_name: 9n_p
library_construction_protocol: 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.  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.  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).  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.