Supplemental Material <b>Optimized protocol for the multi-omics processing of cryopreserved human kidney tissue</b>

Biobanking of tissue from clinically obtained kidney biopsies for later use with multi-omic and imaging techniques is an inevitable step to overcome the need of disease model systems and towards translational medicine. Hence, collection protocols ensuring integration into daily clinical routines using preservation media not requiring liquid nitrogen but instantly preserving kidney tissue for clinical and scientific analyses are of paramount importance. Thus, we modified a robust single nucleus dissociation protocol for kidney tissue stored snap frozen or in the preservation media RNA<i>later</i>and CellCover. Using porcine kidney tissue as surrogate for human kidney tissue, we conducted single nucleus RNA sequencing with the Chromium 10X Genomics platform. The resulting data sets from each storage condition were analyzed to identify any potential variations in transcriptomic profiles. Furthermore, we assessed the suitability of the preservation media for additional analysis techniques (proteomics, metabolomics) and the preservation of tissue architecture for histopathological examination including immunofluorescence staining. In this study, we show that in daily clinical routines the RNA<i>later</i> facilitates the collection of highly preserved kidney biopsies and enables further analysis with cutting-edge techniques like single nucleus RNA sequencing, proteomics, and histopathological evaluation. Only metabolome analysis is currently restricted to snap frozen tissue. This work will contribute to build tissue biobanks with well-defined cohorts of the respective kidney disease that can be deeply molecularly characterized, opening new horizons for the identification of unique cells, pathways and biomarkers for the prevention, early identification, and targeted therapy of kidney diseases.针对临床获取的肾脏活检组织开展生物样本库（Biobanking）留存，以供后续多组学与成像技术研究使用，是摆脱对疾病模型系统依赖、迈向转化医学的不可或缺的关键步骤。因此，开发可融入日常临床流程的样本采集方案尤为重要：这类方案采用无需液氮的保存液，可即时固定肾脏组织以满足临床与科研分析的需求。据此，我们对适用于快速冷冻保存或经RNA Later（RNA later）及CellCover保存液处理的肾脏组织的稳定单细胞核解离方案进行了优化。我们以猪肾脏组织作为人类肾脏组织的替代模型，采用Chromium 10X Genomics平台完成了单细胞核RNA测序。我们对不同保存条件下得到的数据集进行分析，以识别转录组谱中的潜在差异。此外，我们还评估了该类保存液适配蛋白质组学（proteomics）、代谢组学（metabolomics）等其他分析技术的能力，以及其对维持组织结构以开展包括免疫荧光染色（immunofluorescence staining）在内的组织病理学检查的效果。本研究证实，在日常临床流程中使用RNA Later（RNA later）可高效收集保存完好的肾脏活检组织，并支持通过单细胞核RNA测序、蛋白质组学及组织病理学评估等前沿技术开展后续研究。目前仅代谢组分析仍局限于快速冷冻保存的组织样本。本研究将助力构建针对特定肾脏疾病的明确队列组织生物样本库，实现对样本的深度分子表征，为识别肾脏疾病预防、早期筛查及靶向治疗所需的独特细胞类型、信号通路与生物标志物（biomarkers）开辟全新视野。