Genetic basis of thermal nociceptive sensitivity and brain weight in a BALB/c reduced complexity cross: BALB/cJ vs BALB/cByJ Spinal Cord RNAseq. Genetic basis of thermal nociceptive sensitivity and brain weight in a BALB/c reduced complexity cross: BALB/cJ vs BALB/cByJ Spinal Cord RNAseq

We found BALB/cByJ mice showed enhanced sensitivity on the 53.5°C hot plate and mechanical stimulation in the von Frey test compared to BALB/cJ mice and replicated decreased gross brain weight in BALB/cByJ versus BALB/cJ. We then identified a quantitative trait locus (QTL) on chromosome 13 for hot plate sensitivity (LOD = 10.7; p < 0.001; peak = 56 Mb) and a QTL for brain weight on chromosome 5 (LOD = 8.7; p < 0.001). Expression QTL mapping of brain tissues identified H2afy (56.07 Mb) as the top transcript with the strongest association at the hot plate locus (FDR = 0.0002) and spliceome analysis identified differential exon usage within H2afy associated with the same locus. These data are spinal cord RNAseq to confirm differential expression of H2afy and other candidate genes. Overall design: 16 Male and Female BALB/cJ vs BALB/cByJ (N=4/group) mice underwent pain testing at KUMC (PMID:35088629), were sacrificed, spinal cord tissues collected an preserved in RNAlater. RNA was extracted in RNAlater-preserved tissue using Trizol (Qiagen), ethanol precipitation, filtering columns (Qiagen), DNAse digestion (Qiagen), and elution with RNAse and nucleotide free water (Yazdani et al., 2015) and diluted to 100 ng/ul. RNA library preparation (poly-A selection) and RNA-seq were conducted at the University of Chicago Genomics Facility on an Illumina NovaSEQ6000 using a NovaSEQ SP-100 bp flowcell/reagent cassette. We used the R/Bioconductor package “scruff” to conduct demultiplexing, read alignment, read counting, quality checking and data visualization (Wang et al., 2019). Reads were trimmed for quality using Trimmomatic (Bolger et al., 2014). Trimmed reads were then aligned to the mm10 mouse reference genome (Ensembl) to generate BAM files for alignment using STAR (Dobin et al., 2013). For differential gene analysis in the spinal cord, the featureCounts read summarization program was used to count reads mapping to the “exon” feature in a GTF file obtained from Ensembl (GRCm38). Genes without 10 reads per million in at least 3 samples were excluded from analysis using EdgeR 51, and differential gene expression analysis of normalized counts was conducted using an appropriate design matrix, and reported using the topTable function.

本研究发现，相较于BALB/cJ小鼠，BALB/cByJ小鼠在53.5℃热板试验中表现出增强的痛觉敏感性，且在冯·弗雷试验（von Frey test）的机械刺激检测中呈现一致表型；同时验证了BALB/cByJ小鼠整体脑重量低于BALB/cJ小鼠这一结果。随后，本研究在13号染色体上定位到与热板痛觉敏感性相关的定量性状位点（quantitative trait locus, QTL）（LOD=10.7；p<0.001；峰值位点为56 Mb），并在5号染色体上定位到与脑重量相关的QTL（LOD=8.7；p<0.001）。对脑组织开展表达QTL（eQTL）定位分析显示，H2afy基因（位于56.07 Mb处）是热板痛觉敏感性位点关联最强的核心转录本（错误发现率（false discovery rate, FDR）=0.0002）；剪接组分析则发现，该位点相关的H2afy基因存在外显子（exon）使用差异。本数据集为脊髓RNA测序数据，旨在验证H2afy及其他候选基因的差异表达情况。实验整体设计如下：共16只雌雄兼具的BALB/cJ与BALB/cByJ小鼠，每组4只（N=4/组），这些小鼠在堪萨斯大学医学中心（KUMC）完成痛觉相关测试（文献编号PMID:35088629）后被处死，采集脊髓组织并保存于RNAlater溶液中。总RNA提取流程为：采用Trizol试剂（Qiagen公司）、乙醇沉淀法、Qiagen过滤柱、DNase消化（Qiagen公司）以及无RNA酶和核苷酸的水洗脱（Yazdani等，2015）的方法，从RNAlater保存的组织中提取总RNA，随后将其稀释至100 ng/μL。RNA文库制备（采用聚腺苷酸富集（poly-A selection）策略）及RNA测序（RNA-seq）在芝加哥大学基因组学中心完成，使用Illumina NovaSEQ6000测序平台及NovaSEQ SP-100 bp流动槽/试剂套装。本研究使用R/Bioconductor工具包"scruff"完成双端序列拆分（demultiplexing）、reads比对、reads计数、质量评估及数据可视化工作（Wang等，2019）。使用Trimmomatic工具对reads进行质量修剪（Bolger等，2014）。将修剪后的reads比对至Ensembl注释的mm10小鼠参考基因组，使用STAR工具生成比对BAM文件（BAM file）（Dobin等，2013）。针对脊髓组织的差异基因分析，使用featureCounts reads汇总工具对从Ensembl（GRCm38）获取的GTF注释文件（GTF file）中“外显子（exon）”区域比对上的reads进行计数。使用EdgeR工具过滤掉至少3个样本中每百万reads计数不足10的基因，随后通过合适的设计矩阵对标准化计数数据进行差异基因表达分析，并借助topTable函数输出最终分析结果。