title: Effects Of Simulated Space Conditions On CD4+ T Cells: A Multi Modal Analysis
library_construction_protocol: 21 healthy male volunteers who met the inclusion criteria (see supplementary materials) were selected for this study. Blood samples were collected in 9mL lithium heparin tubes (Vacuette, Greiner Bio-One GmbH, Kremsmunster, Austria). Hematological analysis was performed using a Sysmex XS-800i automated hematology analyzer (Sysmex Corporation, Kobe, Japan). The instrument was calibrated and operated according to the manufacturer's instructions. CD4+ T cells were isolated by negative selection with RosetteSep Human CD4+ T Cell Isolation Kits (StemCell Technologies, Inc. Vancouver, BC, Canada) with a purity of 90 to 95% and according to the manufacturer's instructions. The isolated CD4+ T cells were cryopreserved in heat inactivated fetal bovine serum (HI FBS) with 10% DMSO for later use in the experimental procedures. Flow cytometry analysis was performed to confirm the purity of isolated CD4+ T cells using a gating strategy based on the expression of CD3 and CD4 markers. (see supplementary materials)   Upon thawing, cryopreserved CD4+ T cells were stimulated with an anti-CD3/CD28 stimulation cocktail from StemCell Technologies at a concentration of 1 μg/mL in complete T cell expansion medium (StemCell Technologies, Inc. Vancouver, BC, Canada). The cells were then divided into experimental groups. Stimulated CD4+ T cells were incubated at 37 °C with 5% CO2 for 24 h to allow for maximum activation and cytokine secretion, without compromising on cell viability. Figure 2 provides a schematic overview of the experimental design.  Total RNA was extracted from isolated CD4+ T cells using the RNeasy Plus Mini Kit (Qiagen, Germany), according to the manufacturer's protocol. RNA integrity was assessed using the Agilent RNA 6000 Pico Kit and 2100 BioAnalyzer (Agilent Technologies Inc., Santa Clara, CA, USA). The RNA integrity number (RIN) values are shown in the Supplementary materials. For all samples, the RIN values met the requirements for high-quality RNA-sequencing analysis.  Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first-strand cDNA was synthesized using random hexamer primers, followed by second strand cDNA synthesis using either dUTP for the directional library or dTTP for the non-directional library. The non-directional library was prepared after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The directional library was prepared after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time polymerase chain reaction (RT-PCR) for quantification and a bioanalyzer for size distribution detection.