Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies

This paper provides a laboratory workflow for single-nucleus RNA-sequencing (snRNA-seq) including a protocol for gentle nuclei isolation from fresh frozen tumor biopsies, making it possible to analyze biobanked material. To develop this protocol, we used non-frozen and frozen human bladder tumors and cell lines. We tested different lysis buffers (IgePal and Nuclei EZ) and incubation times in combination with different approaches for tissue and cell dissection: sectioning, semi-automated dissociation, manual dissociation with pestles, and semi-automated dissociation combined with manual dissociation with pestles. Our results showed that a combination of IgePal lysis buffer, tissue dissection by sectioning, and short incubation time was the best conditions for gentle nuclei isolation applicable for snRNA-seq, and we found limited confounding transcriptomic changes based on the isolation procedure. This protocol makes it possible to analyze biobanked material from patients with well-described clinical and histopathological information and known clinical outcomes with snRNA-seq.

本研究提出了一套适配单细胞核RNA测序（single-nucleus RNA-sequencing，snRNA-seq）的实验室流程，其中包含了从新鲜冷冻肿瘤活检组织中温和分离细胞核的实验方案，使得对生物样本库储存样本的分析成为可能。为开发该方案，我们使用了非冷冻与冷冻的人膀胱肿瘤组织及细胞系开展实验。我们测试了不同裂解缓冲液（IgePal与Nuclei EZ）及孵育时长，并结合多种组织与细胞解离策略：切片法、半自动解离法、研磨棒手动解离法，以及半自动解离与研磨棒手动解离联用的方法。实验结果表明，采用IgePal裂解缓冲液、组织切片解离法结合短时长孵育的组合方案，是适用于snRNA-seq的最优温和细胞核分离条件；且该分离流程所引入的混淆性转录组变化极为有限。本实验方案可实现对临床与组织病理学信息详实、且已知临床结局的患者来源的生物样本库储存样本进行snRNA-seq分析。