Transcriptomic profiling of nuclei from PFA-fixed and FFPE brain tissues

Formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of clinically annotated specimens, and FFPE-compatible single cell gene expression workflow had been developed and applied recently. However, for tissues where cells are hard to dissociate or brains with complex neuronal cells, nuclear transcriptomic profiling are desirable. Moreover, the effects of standard pathological practice on the transcriptome of samples obtained from such archived specimens was also largely anecdotal. In this study, we reported whole transcriptome sequencing of mouse hippocampus nuclei derived from PFA fixed and FFPE samples, aiming to reveal the effects of PFA fixation, paraffin embedding, fixation time, and FFPE sample preservation time on nuclear mRNA integrity and transcriptome level. In brief, we dissociated the nuclei from PFA fixed for 1 day [PFA(1d)], 3 days [PFA(3d)], 7 days [PFA(7d)] and 15 days [PFA(15d)], and embedded hippocampus for 1 week [FFPE(1w)] and 1 year [FFPE(1y)] and bulk RNA-seq of nuclei was performed as well, taking RNA-seq of fresh frozen (FF(0h)) hippocampus nuclei as criteria.

福尔马林固定石蜡包埋（Formalin-fixed and paraffin-embedded, FFPE）组织档案库是目前规模最大的临床注释标本储备库，近期已开发并应用适配FFPE样本的单细胞基因表达工作流程。然而，对于细胞解离难度较高的组织，或是神经元细胞构成复杂的脑组织，核转录组分析是更为理想的研究手段。此外，标准病理操作流程对这类存档样本转录组特征的影响，目前大多仍缺乏系统实证支撑，多为传闻性结论。
本研究针对经多聚甲醛（Paraformaldehyde, PFA）固定的FFPE样本来源的小鼠海马细胞核开展全转录组测序，旨在揭示PFA固定、石蜡包埋、固定时长以及FFPE样本保存时长对细胞核mRNA完整性及转录组水平的影响。
简言之，我们分别解离了固定时长为1天[PFA(1d)]、3天[PFA(3d)]、7天[PFA(7d)]及15天[PFA(15d)]的PFA固定样本的细胞核，并将海马组织包埋后分别保存1周[FFPE(1w)]与1年[FFPE(1y)]；同时对上述样本的细胞核进行批量RNA测序（bulk RNA-seq），并以新鲜冷冻（FF(0h)）海马核的RNA测序结果作为参照标准。