High-Resolution RNA Sequencing from PFA-Fixed Microscopy Sections
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:2616 |
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| Enthalten in: |
Methods in molecular biology (Clifton, N.J.) - 2616(2023) vom: 30., Seite 205-212 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Ji, Hao [VerfasserIn] |
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| Links: |
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| Themen: |
63231-63-0 |
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| Anmerkungen: |
Date Completed 01.02.2023 Date Revised 05.03.2023 published: Print Citation Status MEDLINE |
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| doi: |
10.1007/978-1-0716-2926-0_16 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 520 | |a 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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