DBiT-seq – in-tissue barcoding for spatially revolved whole transcriptome sequencing at cellular level

Formalin-fixed paraffin-embedded (FFPE) tissues are the most abundant archivable specimens in clinical tissue banks, but unfortunately incompatible with single-cell level transcriptome sequencing due to RNA degradation in storage and RNA damage in extraction. Yale University researchers developed an in-tissue barcoding approach namely DBiT-seq for spatially revolved whole transcriptome sequencing at cellular level, which required no tissue dissociation or RNA exaction, thus potentially more suited for FFPE samples. Herein, we demonstrated spatial transcriptome sequencing of embryonic and adult mouse FFPE tissue sections at cellular level (25um pixel size) with high coverage (>1,000 genes per pixel). Spatial transcriptome of a E10.5 mouse embryo identified all major anatomical features in the brain and abdominal region. Integration with single-cell RNA-seq data for cell type identification indicated that most tissue pixels were dominated by single-cell transcriptional phenotype. Spatial mapping of adult mouse aorta, atrium, and ventricle tissues identified the spatial distribution of different cell types. Spatial transcriptome sequencing of FFPE samples at cellular level may provide enormous opportunities in a wide range of biomedical research. It may allow us to revisit retrospectively the huge resource of clinical tissue specimens to study human disease mechanisms for the discovery of tissue biomarkers and therapeutic targets.

Workflow of DBiT-seq on FFPE samples

(a) Scheme of DBiT-seq on FFPE samples. FFPE tissue blocks stored at room temperature were sectioned into thickness of ~5-7 µm and placed onto a poly-L-lysine coated glass slide. Deparaffinization, rehydration, permeabilization and post-fixation were performed sequentially before placing the 1st PDMS chip on the tissue section. Barcodes A1-A50 were flowed into the microchannels and the reverse transcription was carried out inside each channel. After washing, the 1st PDMS was removed and a 2nd PDMS chip with channels of perpendicular direction was attached on the tissue slide. Ligation reaction mix along with DNA Barcodes B1-B50 were pulled through each of the 50 channels by vacuum and incubated for 30 minutes to perform in situ ligation. Afterwards, the tissue section was digested with Proteinase K to collect cDNA for the downstream processes including template switch, PCR amplification, and tagmentation for NGS library preparation. (b) Deparaffinization of a E10 mouse embryo tissue. It maintained the original morphology with higher contrast after deparaffinization and the fine tissue features were readily discernable. (c) Deformation of tissue section after two sequential microfluidic flows of DBiT-seq. (d) Comparison of gene and UMI counts of our DBiTseq data from FFPE samples with those obtained from other methods including Slide-seq, SlideseqV2 and previous DBiT-seq data from PFA-fixed mouse embryo samples.