title: GSM5792331: Z3 at R-0; Homo sapiens; miRNA-Seq
library_construction_protocol: sEVs were isolated from peripheral blood (PB) samples of 14 astronauts at L-10, R-0, and R+3 using the ExoQuick Plasma preparation and sEV precipitation kit (Cat # EXOQ5TM, System Biosciences, CA, USA). 400 μl of plasma was mixed with thrombin and kept at room temperature (RT) for 5 minutes. Subsequently, samples were centrifuged at 10,000 rpm for 5 minutes. Samples were then incubated with the exosome precipitation solution and refrigerated at 4ºC for 30 minutes. After centrifugation at 1,500 x g for 30 minutes at 4ºC, a beige-colored pellet was observed and dissolved in 100 μl of sterile 1xPBS. NS-300 Nanosight instrument (Malvern Instruments Ltd., Malvern, UK) equipped with an sCMOS camera (Hamamatsu Photonics, Hamamatsu, Japan) and a 405 nm laser was used. Data acquisition and processing were performed using NTA software version 2.3 build 0025. Background extraction was applied, and automatic settings were employed to determine the minimum expected particle size, minimum track length, and blur settings. Data were obtained at camera level 12 (shutter: 600, gain: 350). Three movies of 30 seconds at 25 frames per second were recorded and assigned a single measurement in triplicates. Total RNA from sEVs was isolated from PB samples using miRNeasy kit following manufacturer's protocol (Cat#217084, Qiagen). RNA quality was assessed using an Agilent TapeStation (Agilent, Palo Alto, CA, USA), and RNA concentration was quantified by Qubit 4.0 spectrophotometer. The library for small RNA-sequencing (RNA-Seq) was prepared using the Smarter smRNAseq kit for Illumina (Takara Bio Inc., USA). The quantity and quality of amplified libraries were evaluated using Qubit (Invitrogen, Carlsbad, CA, USA) and Agilent TapeStation high sensitivity D1000 Screen Tape. Small RNA-seq libraries were sequenced using single-end 75 base pairs (SE75) sequencing chemistry on NextSeq 500 instruments following the manufacturer's protocols (Illumina).