Supplemental materials for "Diploid and triploid chinook salmon (<em>Oncorhynchus tshawytscha</em>) have altered microRNA responses in immune tissues after infection with <em>Vibrio</em> <em>anguillarum</em> "

Supplemental files for study investigating the impact of <em>Vibrio</em> <em>anguillarum</em> infection on diploid and triploid Chinook salmon (<em>Oncorhynchus tshawytscha</em>) was investigated to identify if there was any significant immune regulation by microRNAs (miRNA). Small RNAs from hindgut (hg), head kidney (hk), and spleen (sp) were sequenced to determine if miRNA transcript abundance was altered due to ploidy and infection in nine-month old full-sibling diploids and triploids. MiARma-seq and bowtie1 were utilized to align reads to the Chinook salmon genome, and then read abundance associated with miRNA genes quantified using FeatureCounts. Read counts were then analyzed using DESeq2 to identify differentially expressed miRNA between ploidies as well as after infection in all three tissues. Finally, targets of differentially expressed miRNA were predicted using miRanda and pathway impacts predicted using KEGG analysis. <br> Files included: <strong>run_log.log</strong> - miARma-seq logfiles for hindgut (hg), head kidney (hk), and spleen (sp) <strong>stats.log</strong> - miARma-seq stats information for hindgut (hg), head kidney (hk), and spleen (sp) <strong>miarma.cadonic.known.tshawyt.sample.ini</strong> - initiation file for miARma-seq which includes all parameters that were utilized to analyze raw reads <strong>deseq2_cadonic_sample.R</strong> - sample of R code for DEseq2 used to identify differentially expressed miRNA genes <strong>Sasa_Ensembl_3UTR_clean.fasta </strong>- File of 3' untranslated region (UTR) for annotated genes in Ensembl biomart (Ensembl release 105) that was analyzed with miRanda (version v3.3a) to predict miRNA-mRNA relationships. Sequences with unavailable UTRs were cleaned by replacing empty spaces with N using "sed -e '/^[^&gt;]/s/[^ATGC]/N/g' Sasa_Ensembl_3UTR.txt &gt; Sasa_Ensembl_3UTR_clean.fasta". <strong>cadonic_2023_Bioinformatics_protocol_extended.docx - </strong>Extended explanation for the bioinformatic steps and justification for the workflow used in the study.本研究配套补充文件旨在探究**鳗弧菌（*Vibrio anguillarum*）**感染对二倍体与三倍体奇努克鲑（*Oncorhynchus tshawytscha*）的影响，核心目标为明确微小RNA（microRNAs, miRNA）是否介导了显著的免疫调控过程。研究人员针对9月龄全同胞二倍体与三倍体奇努克鲑的后肠（hindgut, hg）、头肾（head kidney, hk）及脾脏（spleen, sp）提取小RNA并开展测序，以解析不同倍性与感染状态下miRNA转录丰度的变化情况。实验分析流程如下：首先使用miARma-seq与bowtie1将测序读段比对至奇努克鲑参考基因组，随后通过FeatureCounts定量miRNA基因对应的读段丰度；继而利用DESeq2对读段计数数据进行差异表达分析，以鉴定不同倍性组间以及感染后三组组织中的差异表达miRNA；最终通过miRanda预测差异表达miRNA的靶基因，并借助KEGG分析预测其通路调控影响。 本数据集包含以下文件： 1. **run_log.log**：后肠、头肾及脾脏样本的miARma-seq运行日志文件 2. **stats.log**：后肠、头肾及脾脏样本的miARma-seq统计信息文件 3. **miarma.cadonic.known.tshawyt.sample.ini**：miARma-seq初始化配置文件，包含分析原始测序读段所用的全部参数 4. **deseq2_cadonic_sample.R**：用于鉴定差异表达miRNA基因的DESeq2示例R代码 5. **Sasa_Ensembl_3UTR_clean.fasta**：经清洗处理的Ensembl生物库（Ensembl 105版本）注释基因3'非翻译区（UTR）序列文件，使用miRanda（v3.3a版本）开展miRNA-mRNA互作预测。该文件通过以下命令完成清洗："sed -e '/^[^>]/s/[^ATGC]/N/g' Sasa_Ensembl_3UTR.txt > Sasa_Ensembl_3UTR_clean.fasta"，将缺失UTR序列的空位替换为N以优化序列格式。 6. **cadonic_2023_Bioinformatics_protocol_extended.docx**：本研究所用生物信息学分析步骤的详细阐释与流程依据说明。