Space ranger pipeline v2022

Our goal in producing Visium CytAssist data was to obtain whole transcriptome, spatially-barcoded sequence data in serial sections. The histology workflow was performed using the Visium CytAssist Spatial Gene Expression for FFPE (Demonstrated Protocol CG000520). The tissue was sectioned as described in Visium CytAssist Spatial Gene Expression for FFPE – Tissue Preparation Guide (Demonstrated Protocol CG000518). 5 µm sections were placed on a Superfrost™ Plus Microscope Slide (Fisherbrand™) and H&E-stained following deparaffinization. Sections were imaged, decoverslipped, followed by hematoxylin destaining and decrosslinking (Demonstrated Protocol CG000520). The glass slide with tissue section was processed with a Visium CytAssist instrument to transfer analytes to a Visium CytAssist Spatial Gene Expression slide with a 0.42 cm² capture area. The probe extension and library construction steps follow the standard Visium for FFPE workflow outside of the instrument. Libraries were sequenced with paired-end dual-indexing (28 cycles Read 1, 10 cycles i7, 10 cycles i5, 90 cycles Read 2). Sequencing libraries were demultiplexed with bcl2fastq (Illumina). The Space Ranger pipeline v2022.0705.1 (10x Genomics) and the GRCh38-2020-A reference were used to process FASTQ files.

Formalin-fixed, paraffin-embedded (FFPE) samples passing the RNA quality control (DV200 > 50%) were used for spatial transcriptomic construction and sequencing. The tissue was prepared according to the Visium CytAssist Spatial Gene Expression for FFPE-Tissue Preparation Guide (CG000518, 10× Genomics, Pleasanton, CA, USA). The sequencing was performed by the Research Institute for Microbial Diseases at Osaka University. Libraries were sequenced on an MGI DNBSEQ-G400RS (MGI Tech Co., Shenzhen, China). The Space Ranger pipeline v2022.0705.1 (10× Genomics, Pleasanton, CA, USA) and the GRCh38-2020-A reference were used to process FASTQ files. The sequence results were guaranteed to be accurate as follows:

Number of spots under tissue: 4397; mean reads per spot: 74,832; median genes per spot: 7195; number of reads: 329,038,054; valid barcodes: 94.0%; valid UMIs: 99.9%; sequencing saturation: 48.0%

UMAP and violin plots were run and plotted using Loupe Browser (10× genomics, Pleasanton, CA, USA). Trajectory analysis and pathway enrichment analysis were performed and plotted using Partek flow software (Partek Incorporated, Chesterfield, MO, USA).