High-Resolution RNA Sequencing from PFA-Fixed Microscopy Sections

• DOI: 10.1007/978-1-0716-2926-0_16
• Publication Date: 2023-01-30T11:04:28Z
• Authors: Hao Ji; Simon Besson-Girard; Peter Androvic; Buket Bulut; Lu Liu; Yijing Wang; Ozgun Gokce
• Keywords: Mice; Microscopy; Sequence Analysis, RNA; Gene Expression Profiling; Animals; RNA; High-Throughput Nucleotide Sequencing; RNA, Messenger

Obtaining high-quality RNA sequencing results from archived biological tissues, such as paraformaldehyde (PFA)-fixed sections for microscopy, is challenging due to the incompatibility of current high-throughput RNA sequencing methods. Here, we present a low-input method for RNA sequencing from archived PFA-fixed sections. Using this method, we routinely obtain high-quality sequencing results from archived mouse brain sections that are prepared for imaging without any special care for avoiding RNA degradation. The PFA cross-linking locks and protects RNA from degradation but cross-linking is also hard to reverse. For this goal, we developed an effective decrosslinking protocol based on Proteinase K activity to retrieve PFA-cross-linked mRNAs which was followed up by a Smart-seq2 library preparation protocol. Our protocol enables spatially defined transcriptomic analysis of archived sections and allows the genomic analysis of PFA-fixed samples. Furthermore, our protocol inactivates pathogenic samples and allows working under regular biosafety levels.