Spatially resolved transcriptomic profiling of degraded and challenging fresh frozen samples. 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. Overall design: 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，其性能表现依赖于高质量RNA样本的储备。此外，当前商用空间转录组检测技术成本高昂，需通过严谨的样本筛选流程提升获取高质量数据的可能性。诚然，由于样本处理、储存及内在生物学因素的影响，RNA质量的前期检测结果可存在显著差异。本研究提出RNA Rescue空间转录组学（RNA-Rescue Spatial Transcriptomics, RRST），这是一款专为改善中低RNA质量新鲜冷冻（fresh frozen, FF）样本的mRNA回收率而开发的空间转录组学工作流程。首先，我们以小鼠脑与前列腺癌样本作为高质量RNA样本的代表，将RRST与标准Visium空间基因表达实验方案开展基准对比测试。随后，我们针对五类极具挑战性的组织类型（包括人肺、结肠、小肠、儿童脑肿瘤以及小鼠骨/软骨）的组织切片，验证了RRST实验方案的适用性。本研究共分析了52个组织切片，结果表明RRST是一款适用于不同质量与来源的新鲜冷冻样本的通用、高效且可重复的实验方案。总体实验设计：针对降解样本或挑战性样本所生成的空间转录组学数据的基因表达谱分析。