SnFFPE-Seq: towards scalable single nucleus RNA-Seq of formalin-fixed paraffin-embedded (FFPE) tissue

Abstract

Profiling cellular heterogeneity in formalin-fixed paraffin-embedded (FFPE) tissues is key to characterizing clinical specimens for biomarkers, therapeutic targets, and drug responses. Here, we optimize methods for isolating intact nuclei and single nucleus RNA-Seq from FFPE tissues in the mouse brain, and demonstrate a pilot application to a human clinical specimen of lung adenocarcinoma. Our method opens the way to broad applications of snRNA-Seq to archival tissues, including clinical samples.

Impact of the Human Cell Atlas on medicine

“…For effective deployment in real-world settings, lab methods need to be sufficiently cost-effective and robust to empower screening and enable adoption, including in under-resourced areas. Connections between the lab and the clinic also need to be further enhanced, including building more biobank resources with rich metadata, large-scale profiling of samples from clinically annotated and diverse cohorts, and better experimental methods to tap into banked samples, especially formalin-fixed paraffin-embedded issues, which are still incompatible with many single-cell methods 133,134 . Among the key computational challenges are the need for open data that reflect human diversity for training computational models, while appropriately safeguarding patient privacy; methods to decode cellular dynamics from static snapshots; algorithms and platforms for efficient querying for genes, cell states and cell types of interest; and fast iterations between lab and computation to design faithful human-derived organoids and cells for screens and therapies.…”

snPATHO-seq: unlocking the pathology archives

“…Clinical samples are routinely preserved as formalin-fixed paraffin-embedded (FFPE) tissue of proven utility in histology and bulk DNA or RNA studies 1,2 . However, exploration of single-cell genomics methods enabling profiling FFPE samples is nascent 2–7 .…”

Single-cell and single-nuclei RNA sequencing as powerful tools to decipher cellular heterogeneity and dysregulation in neurodegenerative diseases

“…Similarly, it is highly recommended to perform snRNA-seq instead of scRNA-seq on samples obtained from formalin-fixed tissues since the fixation process often damages the integrity of various cellular structures, leading to the detection of heavily degraded cytoplasmic RNA ( Esteve-Codina et al, 2017 ). Furthermore, very recent protocols have optimized the extraction of cell nuclei for sequencing from formalin-fixed tissues, including brain ( Chung et al, 2022 ; Vallejo et al, 2022 ). An alternative sample source is in vitro differentiation of either embryonic stem cells (ESCs) or reprogrammed induced pluripotent stem cells (iPSCs), which are a reliable source of biological material since the processes of artificial induction to the desired cell lineage highly recapitulates the molecular processes that occur in the organism during the development and maturation of cells ( Marei et al, 2017 ).…”