Radiation enhances melanoma response to immunotherapeutic and synergizes with benzodiazepines to promote improved anti-tumor activity. Radiation enhances melanoma response to immunotherapeutic and synergizes with benzodiazepines to promote improved anti-tumor activity

The RNA-Seq data of 17 brain metastatic samples short-read sequences were aligned to the hg19 human reference genome using STAR (v 2.4.1a). featureCounts was used to count the reads of the mapped bam files. SAMseq was used to conduct differential expression analysis among the two treatment groups. SAMseq was utilized as it accounts for potential correlation in expression among genes and its permutation-based testing method was deemed more appropriate for a smaller sample size. After identifying the differentially expressed genes (FDR cut-off, 0.05), expression levels were normalized with the samr R package before being log2 transformed. This data was utilized to generate a heatmap with the patients sorted with supervised clustering based upon their immunotherapy/radiation groups and the gene dendrogram was created with Pearson’s Correlation Coefficient and with the Ward D2 linkage method. Differentially expressed genes (FDR<0.05[KD1] ) were subjected to pathway analysis using MetaCore. Overall design: Differential expression analysis across two different patient groups based upon their timing of immunotherapy and radiation therapy.

本数据集涵盖17份脑转移样本的短读长RNA测序（RNA-Seq）数据，使用STAR（v2.4.1a）将测序序列比对至hg19人类参考基因组。采用featureCounts工具对比对生成的BAM格式文件进行读段计数。通过SAMseq方法开展两组处理间的差异表达分析：选用SAMseq是因其可考量基因间表达的潜在相关性，且其基于置换的检验方案更适配小样本量的研究场景。在筛选得到差异表达基因（错误发现率FDR阈值为0.05）后，使用samr R包对基因表达水平进行标准化处理，随后完成对数2转换。本数据集被用于绘制热图：患者样本依据其免疫治疗/放射治疗分组进行监督聚类排序，基因聚类树则基于皮尔逊相关系数与Ward D2聚类连接法构建。将错误发现率（FDR）<0.05的差异表达基因提交至MetaCore进行通路分析。实验整体设计：基于患者免疫治疗与放射治疗的实施时机差异，对两类患者群体开展差异表达分析。