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The spaceflight mice were then euthanized and their eyes were removed and prepared for analysis. All mice received the same ad libitum access to food and water. Animal experiments were approved by the Institutional Animal Care and Use Committee of the University of Tsukuba, Explora Biolabs, JAXA and NASA. No protocol was required from The Loma Linda University (LLU) Institutional Animal Care and Use Committee (IACUC), since only archived frozen and fixed tissues were obtained after euthanasia. 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Trimmed reads were aligned to the Mus musculus + ERCC STAR reference with STAR (version 2.7.10a) and aligned reads were assessed for strandedness using RSeQC Infer Experiment (version 4.0.0) then aligned reads from all samples were quantified using RSEM (version 1.3.1), with strandedness set to reverse. Quantification data was imported to R (version 4.1.3) with tximport (version 1.22.0) and normalized with DESeq2 (version 1.34.0) median of ratios method. Normalized gene counts were subject to differential expression analysis. Differential expression analysis was performed in R (version 4.1.3) using DESeq2 (version 1.34.0); all groups were compared pairwise using the Wald test and the likelihood ratio test was used to generate the F statistic p-value. Gene annotations were assigned using the following Bioconductor and annotation packages: STRINGdb (v2.8.4), PANTHER.db (v1.0.11), and org.Mm.eg.db (v3.15.0).","name":"Sequence Analysis Data Transformation","parameters":[{"@id":"#139820176921312","parameterName":{"@id":"lwanqOYhlZ3TgheWeidAVorCRpt0kYAfJlfldbj0","annotationValue":"User Submitted Raw Counts Tables","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139820176922176","parameterName":{"@id":"ohLPWNgBX1m1IDAO5bT1zOActvuC0uXohHZWildF","annotationValue":"User Submitted Normalized Counts Data","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139820176922848","parameterName":{"@id":"OCCuF0zlUBL2M6BMndL84UhzLQtlkfIpwcEbWOFd","annotationValue":"User Submitted differential expression analysis data","comments":[],"termAccession":"http://purl.obolibrary.org/obo/OBI_0002584","termSource":"OBI"}}],"protocolType":{"@id":"dYomwZpWQToAk3GGMavveUBJ66aoAqbWbu7JSna0","annotationValue":"Sequence Analysis Data Transformation","comments":[],"termAccession":"","termSource":"MESH"},"uri":"","version":""},{"@id":"#139818430947632","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":"#139820162492832","parameterName":{"@id":"Ohs1BbENFJo4CWnwszKmpZ4g1nZJNqqJv60k0wyP","annotationValue":"Read Depth","comments":[],"termAccession":"http://ncicb.nci.nih.gov/xml/owl/EVS/Thesaurus.owl","termSource":"NCIT"}},{"@id":"#139820162492304","parameterName":{"@id":"fGrXPRyXNbGVvShodvkdxxY2geN1g064LhQ56lig","annotationValue":"rRNA estimation","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139820162493408","parameterName":{"@id":"ahid9PluC4PDyLrGsFf76ktvkDjjMpBswqDNjOZA","annotationValue":"MultiQC Data Files","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":"#139818430741232","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) concatenated with ERCC92.fa from ThermoFisher (<a href=\"https://assets.thermofisher.com/TFS-Assets/LSG/manuals/ERCC92.zip\" target=\"_\" >https://assets.thermofisher.com/TFS-Assets/LSG/manuals/ERCC92.zip</a>), and the following gtf annotation file: Mus_musculus.GRCm39.112.gtf concatenated with the ERCC92.gtf file from ThermoFisher (<a href=\"https://assets.thermofisher.com/TFS-Assets/LSG/manuals/ERCC92.zip\" target=\"_\" >https://assets.thermofisher.com/TFS-Assets/LSG/manuals/ERCC92.zip</a>). Trimmed reads were aligned to the Mus Musculus + ERCC 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), ERCC genes were removed, and the non-ERCC genes were 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. Gene annotations were assigned using the custom annotation tables generated in-house as detailed in GL-DPPD-7110-A (<a href=\"https://github.com/nasa/GeneLab_Data_Processing/blob/GL_RefAnnotTable-A_1.1.0/GeneLab_Reference_Annotations/Pipeline_GL-DPPD-7110_Versions/GL-DPPD-7110-A/GL-DPPD-7110-A.md\" target=\"_\" >https://github.com/nasa/GeneLab_Data_Processing/blob/GL_RefAnnotTable-A_1.1.0/GeneLab_Reference_Annotations/Pipeline_GL-DPPD-7110_Versions/GL-DPPD-7110-A/GL-DPPD-7110-A.md</a>), with STRINGdb (version 2.16.4), PANTHER.db (version 1.0.12), and org.Mm.eg.db (version 3.19.1). Assessment of ERCC spiked genes was performed as detailed in the ERCC_analysis_GLbulkRNAseq.html report.","name":"GeneLab RNAseq data processing protocol","parameters":[{"@id":"#139818502867312","parameterName":{"@id":"W9tes96RPBkIuuA0eNexPPFOXF4m5lLPOIBTnVsM","annotationValue":"trimmed sequence data","comments":[],"termAccession":"http://purl.obolibrary.org/obo/OBI_0002569","termSource":"OBI"}},{"@id":"#139818502865584","parameterName":{"@id":"s2HskUjUzSQmahcRYDfv1xBVjYScvewE8EO773hd","annotationValue":"Trimmed Sequence Data/Trimming Reports","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502867408","parameterName":{"@id":"Paz4Pjo6sV6E66RG3a9xuZDjKKpk7ywwq3VwmAwn","annotationValue":"Trimmed Sequence Data/MultiQC Reports","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502866832","parameterName":{"@id":"lqpWRJZ7O2rPhdA4uPNANCqeFig4cQhw07RfB2f5","annotationValue":"aligned sequence data","comments":[],"termAccession":"http://purl.obolibrary.org/obo/OBI_0002580","termSource":"OBI"}},{"@id":"#139818502868224","parameterName":{"@id":"SPhWEr1D3r2VJ9OKO5b94MEY02Ipod6UwkLtQaRk","annotationValue":"Aligned Sequence Data/Unmapped Reads","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502865056","parameterName":{"@id":"3AwnKZQDUPRaMntinM9bvBrGHpnb8qc6GaaZRIQh","annotationValue":"Aligned Sequence Data/Alignment Logs","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502868848","parameterName":{"@id":"ib7AEGeVUoAruCpz91Qn6GXdO3E5d8k0DSeT2EQR","annotationValue":"Aligned Sequence Data/MultiQC Reports","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502866400","parameterName":{"@id":"QcbiokEiY9OO2PO8IXfNhCwNUeMq4JM2ugyguLO3","annotationValue":"RSeQC/MultiQC Reports","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502867792","parameterName":{"@id":"CzV3xNixKDCRnULWE97WbT9v456v77sgf4dPZG0Y","annotationValue":"Raw Counts Data","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502867744","parameterName":{"@id":"WXhsQG8JLZ5oK8kUYbPxGSfXi520yz8A4dEHcBKs","annotationValue":"Raw Counts Tables","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502867648","parameterName":{"@id":"w31o7Q91WYOVYIwRCqOCvdUs4H84WG2M5y8Qgu1N","annotationValue":"Raw Counts Data/MultiQC Reports","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502866064","parameterName":{"@id":"Lns3jxVPjBQ2dUuFO7KyPzSqST1hCHcuYbzwld4i","annotationValue":"Normalized Counts Data","comments":[],"termAccession":"https://osdr.nasa.gov/","termSource":""}},{"@id":"#139818502866688","parameterName":{"@id":"c4UoiNar4sA4ty0VawH08rhaf4AR7F8drWBP91tt","annotationValue":"differential expression analysis data","comments":[],"termAccession":"http://purl.obolibrary.org/obo/OBI_0002584","termSource":"OBI"}},{"@id":"#139818502868512","parameterName":{"@id":"RU82pDdGEcbsiezMzkaKgjf1MKkk9FaQCjynpZUS","annotationValue":"ERCC Analyses","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":""}],"publicReleaseDate":"06-Nov-2024","publications":[],"studyDesignDescriptors":[],"submissionDate":"12-Aug-2024","title":"Artificial Gravity Attenuates the Transcriptomic Response to Spaceflight in the Optic Nerve and Retina -- Optic nerve data","unitCategories":[{"@id":"0egjOe4tupLenTJ2RAdBhRffOpl8xZWv0FRLbF9W","annotationValue":"day","comments":[],"termAccession":"http://purl.obolibrary.org/obo/UO_0000033","termSource":""},{"@id":"ixhZXi79C7bGPbpBgn0nhYS3WcKUHFxvfrXat1ju","annotationValue":"degree Celsius","comments":[],"termAccession":"http://purl.obolibrary.org/obo/UO_0000027","termSource":""},{"@id":"BFtmHv7jE1g1SEfuffoEIGHvLtR5icFOjFbLPFs8","annotationValue":"gram","comments":[],"termAccession":"http://purl.obolibrary.org/obo/UO_0000021","termSource":""},{"@id":"1elCZM8nktXBnEgzvNbC6OfVMVRCjhLVI3KdVQv4","annotationValue":"week","comments":[],"termAccession":"http://purl.obolibrary.org/obo/UO_0000034","termSource":""}]}],"version":5}},"success":true}