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. 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℃热板实验中表现出增强的痛觉敏感性，且在冯·弗雷（von Frey）机械刺激实验中呈现相似的痛觉反应变化；同时验证了BALB/cByJ小鼠的全脑重量较BALB/cJ小鼠有所降低。随后，我们在13号染色体上定位到一个与热板痛觉敏感性相关的数量性状位点（quantitative trait locus, QTL）（LOD=10.7；p<0.001；峰值位点位于56 Mb处），并在5号染色体上定位到一个与脑重量相关的QTL（LOD=8.7；p<0.001）。对脑组织开展的表达数量性状位点（expression QTL, eQTL）定位分析显示，H2afy（位于56.07 Mb处）是热板痛觉敏感性位点中关联性最强的核心转录本（错误发现率FDR=0.0002）；剪接组分析则发现，H2afy基因内存在与该位点相关的外显子差异使用现象。本数据集为脊髓RNA测序（RNA-seq）数据，用于验证H2afy及其他候选基因的差异表达情况。实验共纳入16只雌雄BALB/cJ与BALB/cByJ小鼠（每组n=4），于KUMC完成痛觉行为学测试（PMID:35088629），处死后收集脊髓组织并置于RNAlater保护液中保存。采用TRIzol试剂（Qiagen）对RNAlater保存的组织进行RNA提取，随后通过乙醇沉淀法、过滤柱纯化、DNA酶消化（Qiagen）及无RNA酶无核苷酸水洗脱（Yazdani等，2015）的流程获取总RNA，并将RNA浓度稀释至100 ng/μL。RNA文库制备采用poly(A)富集选择策略，RNA测序工作由芝加哥大学基因组学中心在Illumina NovaSEQ6000测序平台上完成，使用NovaSEQ SP-100 bp流动槽及试剂卡匣进行测序。我们使用R/Bioconductor软件包"scruff"完成解多路复用、读段比对、读段计数、质量质控及数据可视化分析（Wang等，2019）。采用Trimmomatic工具对原始读段进行质量修剪（Bolger等，2014）。将修剪后的读段比对至小鼠参考基因组mm10（Ensembl数据库），并通过STAR比对工具生成比对后的BAM格式文件（Dobin等，2013）。针对脊髓组织的差异基因表达分析，我们采用featureCounts读段汇总工具，对从Ensembl数据库获取的GTF格式文件（GRCm38版本）中"exon"特征区域的比对读段进行计数。过滤掉至少3个样本中每百万读段数少于10的基因，随后使用EdgeR软件包进行数据分析，通过合适的设计矩阵对标准化后的读段计数进行差异基因表达分析，并借助topTable函数输出结果。