(B-47) Highly Sensitive Spatial Omics Sequencing of Formalin-Fixed Paraffin-Embedded Tissue

Introduction::
The emerging technology of formalin-fixed paraffin-embedded (FFPE) spatial transcriptomics offers an extensive understanding of gene expression at the tissue level for archived tissue blocks. However, due to the compromised RNA quality in FFPE tissues, current methodologies for FFPE spatial transcriptomics mostly employ probe-based approaches, resulting in non-comprehensive or biased transcriptomic profiles. In this work, we develop an innovative technique which enables the evaluation of gene expression at the whole-genome level with base pair resolution for FFPE samples using polyadenylation. The utilization of sequencing-based in situ barcoding through microfluidic devices allows for seamless integration with other analytical modalities. Our approach holds significant potential for improving gene capture efficiency as well as conducting multi-omics studies on FFPE samples.




Materials and Methods::
Mouse embryo E11 and mouse lymph node FFPE tissue sections with 10μm thickness were purchased from Zyagen. Deparaffinization and rehydration were performed using xylene and a graded ethanol series, and the antigen retrieval was performed with antigen retrieval buffer (Abcam). Permeabilization, in situ polyadenylation and reverse transcription are performed, followed by two-step ligation with oligos that has spatial barcode flowing through microfluidic device. Then, tissuesections were lysed to retrieve barcoded cDNA. After PCR amplification, rRNA removal and library building, the generated gene expression library was sequenced using Illumina platform. For the data analysis part, raw sequencing data were processed using ST-pipeline for alignment, barcode assignment, and gene expression quantification. Unsupervised clustering and differential expression were analyzed and visualized to identify distinct molecular subtypes and spatial patterns within the tissue samples.




Results, Conclusions, and Discussions:: Results and Discussion: Both deterministic barcoding in tissue (DBiT) and the enhanced DBiT with in situ polyadenylation (DBiT_PAP) were performed on FFPE samples. By selecting a resolution of 50μm and 20μm spatial pixel size, we achieved a highly reproducible high-resolution spatial profiling. Our experimental outcomes demonstrate that the addition of polyA tails using polymerase can effectively rescue degraded or cross-linked mRNA in FFPE specimens. We observed an average increase of 43% in gene counts and a substantial 149% increment of transcripts capture. Furthermore, unsupervised clustering and spatial mapping of transcriptomic profiles, which are congruent with tissue morphology, reveal the underlying biological architecture of the mouse embryo.

Conclusions: The in-situ polyadenylation method effectively recovers degraded RNA lacking polyA tails, to some extent, and presents a more impartial capture of transcriptomic signals in comparison to probe-based methodologies. This approach unlocks the potential for conducting multi-omics spatial investigations on real-world archived tissue samples to dissect molecular determinants underlying complex diseases and various clinical responses.


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