核糖核酸
计算生物学
协议(科学)
转录组
多聚甲醛
降级(电信)
生物
计算机科学
化学
基因
遗传学
基因表达
医学
病理
有机化学
替代医学
电信
作者
Hao Ji,Simon Besson‐Girard,Peter Androvic,Buket Bulut,Lu Liu,Yijing Wang,Özgün Gökçe
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 205-212
被引量:1
标识
DOI:10.1007/978-1-0716-2926-0_16
摘要
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.
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