RNAseq of Mineralocorticoid Receptor (MR) overexpression in UVB-induced ocular rosacea model on meibomian glands of rats

The aim of this study was to analyse the transcriptional regulation of UVB-induced ocular rosacea model on the meibomian glands of rats that overexpress the human NRC32 gene coding for mineralocorticoid receptor (P1.hMR) and age- and sex- matched wild type (WT) littlemates. 10-12 weeks old female Wild-Type (WT) and P1.hMR rats were subjected to 5-days UVB irrdiation on eyelids (UVB_WT: n = 3, UVB_P1.hMR: n = 5), together with age- and sex- matched rats without UVB-induced model (Control_WT: n = 4, Control_P1.hMR: n = 5), they were euthanized and the meibomian glands were dissected for RNA-sequencing. Total RNA samples were sequenced at the iGenSeq transcriptomic platform of the Brain and Spine Institute (ICM, Paris, France). RNA quality was checked by capillary electrophoresis and RNA integrity numbers (RIN) ranging from 8.8 to 9.3 was accepted for library generation. Quality of raw data was assessed using FastQC. Poor quality sequences and adapters were trimmed or removed with fastp, using default parameters, to retain only good quality paired reads. Illumina DRAGEN bio-IT Plateform (v3.8.4) was used for mapping on rn7 reference genome. Library orientation, library composition and coverage along transcripts were checked with Picard tools. Differential analysis has been also conducted with DESeq2. Multiple hypothesis adjusted p-values were calculated with the Benjamini-Hochberg procedure to control FDR. FDR threshold was set at 0.05, log2 fold-change threshold was set at 0.5. We identified genes differentially regulated in UVB-treated WT as compared to Control WT, and genes differentially regulated in UVB-treated P1.hMR rats as compared to Control P1.hMR.本研究旨在分析过表达编码盐皮质激素受体（mineralocorticoid receptor）的人类NRC32基因的P1.hMR大鼠，以及与年龄、性别匹配的同窝野生型（wild type, WT）大鼠的睑板腺UVB诱导性眼酒渣鼻模型的转录调控情况。选取10~12周龄的雌性野生型（WT）及P1.hMR大鼠，对其眼睑实施为期5天的UVB照射（UVB_WT组：n=3；UVB_P1.hMR组：n=5）；同时设置与年龄、性别匹配的未接受UVB造模的对照组（Control_WT组：n=4；Control_P1.hMR组：n=5）。实施安乐死后解剖分离各组大鼠的睑板腺，进行RNA测序（RNA-sequencing）。总RNA样本在法国巴黎脑与脊柱研究所（ICM）的iGenSeq转录组平台完成测序。通过毛细管电泳检测RNA质量，仅选取RNA完整性数值（RNA Integrity Number, RIN）介于8.8至9.3之间的样本用于文库构建。使用FastQC评估原始数据质量，采用fastp工具（默认参数）修剪或移除低质量序列与测序接头，仅保留高质量双端测序读段。使用Illumina DRAGEN bio-IT平台（v3.8.4）将测序读段比对至rn7参考基因组。通过Picard工具检查文库定向性、文库组成及转录本覆盖度。采用DESeq2进行差异表达分析，通过Benjamini-Hochberg法计算多重假设检验校正后的p值以控制错误发现率（False Discovery Rate, FDR），设置FDR阈值为0.05，log2倍变化阈值为0.5。本研究分别鉴定了UVB照射的WT大鼠相较于对照WT大鼠，以及UVB照射的P1.hMR大鼠相较于对照P1.hMR大鼠的差异调控基因。