Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples

Spatially resolved transcriptomics has enabled precise genome-wide mRNA expression profiling within tissue sections. The performance of unbiased SRT methods targeting the polyA tail of mRNA, relies on the availability of specimens with high RNA quality. Moreover, the high cost of currently available SRT assays requires a careful sample screening process to increase the chance of obtaining high-quality data. Indeed, the upfront analysis of RNA quality can show considerable variability due to sample handling, storage, and/or intrinsic factors. We present RNA-Rescue Spatial Transcriptomics (RRST), an SRT workflow designed to improve mRNA recovery from fresh frozen specimens with moderate to low RNA quality. First, we provide a benchmark of RRST against the standard Visium spatial gene expression protocol on high RNA quality samples represented by mouse brain and prostate cancer samples. Then, we demonstrate the RRST protocol on tissue sections collected from five challenging tissue types, including: human lung, colon, small intestine, pediatric brain tumor, and mouse bone/cartilage. In total, we analyzed 52 tissue sections and our results demonstrate that RRST is a versatile, powerful, and reproducible protocol for FF specimens of different qualities and origins. Gene expression profiiles of spatially resolved transcriptomics data generated from degraded or challenging samples空间转录组学（Spatially resolved transcriptomics, SRT）已实现组织切片内全基因组范围的精准mRNA表达谱分析。靶向mRNA polyA尾的无偏空间转录组学方法，其性能依赖于高质量RNA样本的可获得性。此外，当前商用空间转录组学检测成本高昂，需通过严格的样本筛选流程以提升获取高质量数据的概率。诚然，受样本处理、储存及内在因素影响，RNA质量的前期分析结果往往存在显著差异。本研究提出RNA拯救型空间转录组学（RNA-Rescue Spatial Transcriptomics, RRST），这是一种针对RNA质量中等至偏低的新鲜冷冻（fresh frozen, FF）样本、旨在提升mRNA回收率的空间转录组学实验流程。首先，我们以小鼠脑和前列腺癌样本作为高质量RNA样本的代表，将RRST与标准Visium空间基因表达实验流程进行了基准对比。随后，针对人类肺、结肠、小肠、儿童脑肿瘤及小鼠骨/软骨五种具有挑战性的组织类型的切片样本，我们验证了RRST实验流程的可行性。本研究共计分析52份组织切片，结果表明，RRST是一款通用、高效且可重复的实验流程，适用于不同质量与来源的新鲜冷冻样本。从降解样本或挑战性样本中生成的空间转录组学数据的基因表达谱