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All experiments were approved by the Institutional Animal Care and Use Committee of JAXA (Protocol Number: 016-014B for MHU-1, No. 018-011D for MHU-4, and No. 018-036D for MHU-5), Explora Biolabs (Study Number: EB15-010A for MHU-1, No. EB19-003 for MHU-4, and No. SP19-003 for MHU-5), and NASA (Protocol Number: NAS-15-004-Y1 for MHU-1, No. FLT-18-118 for MHU-4, and No. JAXA MHU-5/FLT-19-121 for MHU-5). All experiments were conducted according to the guidelines and applicable laws in Japan and the USA.\n\nMHU-1: Twelve male C57BL/6\u2009J mice (8 weeks old) in the TCU were launched aboard SpX-9 on June 18, 2016, from the NASA Kennedy Space Center (KSC) in Florida and were then transported to the ISS. The mice were divided into two groups: the MG group experienced microgravity, and the AG group was exposed to 1\u2009g artificial gravity on the bottom floor of habitat cage units (HCUs) via centrifugation at 77\u2009rpm in MARS-S (15\u2009cm radius).\n\nMHU-4: Six male C57BL/6\u2009J mice (9 weeks old) in the TCU were launched aboard SpX-17 on May 7, 2019, from the NASA KSC and were then transported to the ISS. The MHU-4_PG group was maintained in an artificial gravity environment at 1/6\u2009g on the bottom floor of the HCU via centrifugation at 31\u2009rpm in MARS-S (15\u2009cm radius).\n\nMHU-5: Six C57BL/6\u2009J male mice (9 weeks old) in the TCU were launched aboard SpX-20 on March 10, 2020, from the NASA KSC and were then transported to the ISS. The MHU-5_PG group was maintained in an artificial gravity environment at 1/6\u2009g on the bottom floor of the HCU via centrifugation at 21\u2009rpm in MARS-L (35\u2009cm radius).\n\nThe GC group comprised six age-matched (8 or 9 weeks old) male C57BL/6\u2009J mice per mission, maintained under the same conditions as those aboard the ISS. All mice from each mission were sacrificed on the second day after returning to Earth.  The left and right Sol and EDL muscles were neatly stacked on top of each other. The Achilles tendon side was vertically mounted on the tragacanth gum on a cork disc and quickly frozen in isopentane cooled in liquid nitrogen. Thin frozen muscle sections (8\u2009\u00b5m) were subjected to immunohistochemical and RNA-seq analyses.\n\nDuring the flight, temperature and humidity data loggers were attached to the TCU (launch and return phase) or inside the centrifuge-equipped biological experiment facility (CBEF-ALT) or CBEF-L (onboard phase) in all three missions. Following launch of the MHU-4 and MHU-5 missions, the logger data were processed. The MHU-1 mission data were previously published. The average temperatures at the onboard phases of MHU-4 and MHU-5 were 23.1C and 23.6C, respectively. Temperatures higher than 30  \u030aC were observed in the return phase of MHU-5, especially in the sea transportation phase, and lasted 16 h. The average relative humidities at the onboard phases were 47.5% and 39.7% for MHU-4 and MHU- 5, respectively. The gas concentrations of carbon dioxide and ammonia were monitored by sensors attached inside the CBEF-ALT or CBEF-L. These concentrations were kept at a low level during the mission. The results indicate that mice were housed under a properly controlled environment. Since the radiation level in space is much higher than that on the ground, the total radiation dose during the approximately one-month flight was measured using the Bio Passive Dosimeter for Life Science Experiments in Space (Bio PADLES). The radiation absorbed dose rate was 0.31 \u00b1 0.02 (MHU-4) and 0.36 \u00b1 0.02 (MHU-5) mGy/day, and the dose equivalent rate was 0.48 \u00b1 0.03 (MHU-4) and 0.79 \u00b1 0.05 (MHU-5) mSv/day during the two space experiments. While high doses of radiation affect the health of mice, these values were comparable to those of the MHU-1 mission and appeared to be low- level, not causing the acute reactions in mice as those observed in astronauts.\n\nThe GC group comprised six age-matched (8 or 9 weeks old) male C57BL/6\u2009J mice per mission, maintained under the same conditions as those aboard the ISS. 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Thin frozen muscle sections (8\u2009\u00b5m) were subjected to immunohistochemical and RNA-seq analyses.","name":"sample collection","parameters":[{"@id":"#139782885264112","parameterName":{"@id":"eUm691aR7an5kII9bYi7JOSQEcghOzzbVfEjAtOG","annotationValue":"Sample Preservation Method","comments":[],"termAccession":"https://genelab-data.ndc.nasa.gov/","termSource":""}},{"@id":"#139782885261520","parameterName":{"@id":"vbrzINCaNhslTc1WulmcigpF6afqM7x3H1PCAdm0","annotationValue":"Sample Storage Temperature","comments":[],"termAccession":"https://genelab-data.ndc.nasa.gov/","termSource":""}},{"@id":"#139782885264544","parameterName":{"@id":"oAsjGOgEqTfWF2wfmtxS6Gcr7hYI1ETT3CEZVbT8","annotationValue":"Age at Euthanasia","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}}],"protocolType":{"@id":"zaKRMPLXOBudyE1ROMNBjnZXViwkiB6PT7wea0yd","annotationValue":"sample collection","comments":[],"termAccession":"","termSource":""},"uri":"","version":""},{"@id":"#139782959074992","comments":[],"components":[],"description":"Thin frozen muscle sections (8\u2009\u00b5m) were subjected to immunohistochemical and RNA-seq analyses.","name":"Tissue Sectioning","parameters":[{"@id":"#139782885262048","parameterName":{"@id":"35PrCwQH0dipXtJtV9CanRRL9UOddg8ArC2DZqE0","annotationValue":"Section Thickness","comments":[],"termAccession":"http://ncicb.nci.nih.gov/xml/owl/EVS/Thesaurus.owl","termSource":"NCIT"}}],"protocolType":{"@id":"RRsPRNYhNG86L6iBmJJlvk4piPzKWvkdGlZLRyNV","annotationValue":"Tissue Sectioning Protocol","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""},"uri":"","version":""},{"@id":"#139782959072688","comments":[],"components":[],"description":"Using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), total RNA was extracted from 100 sections of 8\u2009\u03bcm frozen Sol muscle tissue, following standard protocols.","name":"Nucleic Acid Extraction","parameters":[{"@id":"#139782885263344","parameterName":{"@id":"3Hq6ViAPdhnUQVpmPN4g5qe1Sc2CIbQALaTc3jdF","annotationValue":"","comments":[],"termAccession":"","termSource":""}}],"protocolType":{"@id":"hvmnimkOSUuDnRXoMCmOVO622eV9H3rhB5Orz2Zc","annotationValue":"Nucleic Acid Extraction","comments":[],"termAccession":"","termSource":""},"uri":"","version":""},{"@id":"#139782885263728","comments":[],"components":[],"description":"An RNA-seq library was prepared using the NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) after ribosomal RNA (rRNA) depletion using the NEBNext rRNA Depletion Kit (New England Biolabs).","name":"Library Construction","parameters":[{"@id":"#139782885263200","parameterName":{"@id":"SXpkstgzgf9WOhn8BRUImqf5kbHf1TJ3GjkLiaMg","annotationValue":"Library Selection","comments":[],"termAccession":"","termSource":""}},{"@id":"#139782885263584","parameterName":{"@id":"rlzTK2chbZkUcaCWHWpzFmlVUzKNZ7DXWAzUXMTb","annotationValue":"Library Layout","comments":[],"termAccession":"","termSource":""}},{"@id":"#139782885263296","parameterName":{"@id":"FgvdSWC3uI17LsXvErGgSgUHhsklR2Ok3RnEeJgE","annotationValue":"Stranded","comments":[],"termAccession":"","termSource":""}}],"protocolType":{"@id":"jlgnONtnl1cbnUwKKehnj9aCWkaf8ZPvhHTdzhV8","annotationValue":"Library Construction","comments":[],"termAccession":"","termSource":""},"uri":"","version":""},{"@id":"#139782885261952","comments":[],"components":[],"description":"Paired-end (2\u2009\u00d7\u200936 bases) sequencing was performed using the NextSeq500 platform (Illumina, San Diego, CA, USA).","name":"Nucleic Acid Sequencing","parameters":[{"@id":"#139782885264928","parameterName":{"@id":"QZv3O6FfjrX7Rj1AdVBpE7MMkvX0gDPFNyhTgsul","annotationValue":"Sequencing Instrument","comments":[],"termAccession":"","termSource":""}},{"@id":"#139782885264832","parameterName":{"@id":"CtQ18ML4iL8up3q8jIQxtenGoYSNWw4i3XMQYFwU","annotationValue":"Base Caller","comments":[],"termAccession":"","termSource":""}},{"@id":"#139782895071824","parameterName":{"@id":"DOWz9RyvfMoltYGyC7An2WqtHdVXvLLJU32JiRbM","annotationValue":"Read Length","comments":[],"termAccession":"http://ncicb.nci.nih.gov/xml/owl/EVS/Thesaurus.owl","termSource":"NCIT"}}],"protocolType":{"@id":"vbHeywu5lMpfsh3jNyY18mRWy5aZupc8gVSXD5Uh","annotationValue":"Nucleic Acid Sequencing","comments":[],"termAccession":"","termSource":""},"uri":"","version":""},{"@id":"#139782885265024","comments":[],"components":[],"description":"Fastq format was checked with fastq_utils v0.25.1. Quality assessment of reads was performed with FastQC v0.12.1 and reports were combined with MultiQC v1.26. rRNA percentages of reads were estimated with the 'hts_SeqScreener' program of htstream v1.3.3 screened against a fasta file of Mus-musculus rRNA sequences retrieved from NCBI on 01-Sep-2021.","name":"GeneLab raw data processing protocol","parameters":[{"@id":"#139782895075280","parameterName":{"@id":"Ohs1BbENFJo4CWnwszKmpZ4g1nZJNqqJv60k0wyP","annotationValue":"Read Depth","comments":[],"termAccession":"http://ncicb.nci.nih.gov/xml/owl/EVS/Thesaurus.owl","termSource":"NCIT"}},{"@id":"#139782895071872","parameterName":{"@id":"RaWNd099ZqS9NO9TqfvpU9arANv1Do2iAJueQJMm","annotationValue":"rRNA Contamination","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139782895074896","parameterName":{"@id":"WoqQnOe0wYp5oJdMtNlLbe7bcXgG6P1BgtOCncYd","annotationValue":"MultiQC File Names","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}}],"protocolType":{"@id":"gHRCpX8ZCZrvMb1jv0PpSihU4DurynDUUnkOSfet","annotationValue":"data transformation","comments":[],"termAccession":"http://purl.obolibrary.org/obo/OBI_0200000","termSource":"OBI"},"uri":"","version":""},{"@id":"#139782885261856","comments":[],"components":[],"description":"Data were processed as described in GL-DPPD-7101-G (<a href=\"https://github.com/nasa/GeneLab_Data_Processing/blob/master/RNAseq/Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-G.md\" target=\"_\" >https://github.com/nasa/GeneLab_Data_Processing/blob/master/RNAseq/Pipeline_GL-DPPD-7101_Versions/GL-DPPD-7101-G.md</a>), using NF_RCP version 2.0.2 (https://github.com/nasa/GeneLab_RNASeq_Workflow/tree/NF_RCP_2.0.2). In short, raw fastq files were filtered using Trim Galore! (version 0.6.10) powered by Cutadapt (version 4.2). Trimmed fastq file quality was evaluated with FastQC (version 0.12.1), and MultiQC (version 1.26) was used to generate MultiQC reports. Mus Musculus STAR and RSEM references were built using STAR (version 2.7.11b) and RSEM (version 1.3.3), respectively, Ensembl release 112, genome assembly GRCm39 (Mus_musculus.GRCm39.dna.primary_assembly.fa) and the following gtf annotation file: Mus_musculus.GRCm39.112.gtf. Trimmed reads were aligned to the Mus Musculus STAR reference with STAR (version 2.7.11b) and alignment log files were compiled with MultiQC (version 1.26). Aligned reads were assessed for strandedness using RSeQC infer_experiment.py (version 5.0.4), gene body coverage using RSeQC geneBody_coverage.py (version 5.0.4), inner distance was assessed using RSeQC inner_distance.py (version 5.0.4), and distribution of RNA features using RSeQC read_distribution.py (version 5.0.4). Aligned reads from all samples were quantified using RSEM (version 1.3.3), with strandedness set to reverse and RSEM count log files were compiled with MultiQC (version 1.26). To create a set of rRNA-removed quantification data, Ensembl IDs mapping to ribosomal RNA (rRNA) were removed from the RSEM gene count data. Both the original set of RSEM quantification data and the rRNA-removed data were subject to differential gene expression analysis as follows. A runsheet was generated with dp_tools (version 1.3.8) and the runsheet and quantification data were imported to R (version 4.4.2) with tximport (version 1.34.0) and normalized with DESeq2 (version 1.46.0) median of ratios method. Normalized gene counts were subject to differential expression analysis. Differential expression analysis was performed in R (version 4.4.2) using DESeq2 (version 1.46.0); all groups were compared pairwise using the Wald test and the likelihood ratio test was used to generate the F statistic p-value. 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