Aggregate of Mouse Brain Sections: CytAssist for FFPE

10x Genomics obtained FFPE Mouse Brain tissue blocks from Avaden Biosciences. The tissue sections were sectioned as described in Visium CytAssist Spatial Gene Expression for FFPE – Tissue Preparation Guide Demonstrated Protocol (CG000518). Serial tissue sections of 5 µm were placed on standard glass slides, then H&E-stained following deparaffinization. Sections were coverslipped with 85% glycerol, imaged, decoverslipped, followed by dehydration & decrosslinking Demonstrated Protocol (CG000520). The glass slides with tissue sections were processed via Visium CytAssist instrument to transfer analytes to the same Visium CytAssist Spatial Gene Expression slide. The probe extension and library construction steps follow the standard Visium for FFPE workflow outside of the instrument.

The H&E images of each section were acquired using Olympus VS200 Slide Scanning Microscope with these settings:

• Olympus Objective magnification: 20x UPLXAPO Objective
• Numerical Aperture: 0.8
• ScopeLED light source: Xcite Novum
• Camera: VS-264C
• Exposure: 500 µs

Individual libraries for each section were prepared following the Visium CytAssist Spatial Gene Expression Reagent Kits for FFPE User Guide (CG000495). Sequencing data of the individual sections was first processed using spaceranger count. The outputs were then aggregated and normalized to equal sequencing depth using the spaceranger aggr pipeline to generate a single set of output files containing all of the data from each of the individual samples.

• Sequencing instrument: Illumina NovaSeq, flow cell HNNNMDSX3
• Sequencing configuration: 28bp read 1 (16bp Visium spatial barcode, 12bp UMI), 90bp read2 (transcript), 10bp i7 sample barcode and 10bp i5 sample barcode
• Slide: V42A20-353

For individual section level metrics, refer to the dataset pages:

• Mouse Brain Coronal Section 1 (FFPE)
• Mouse Brain Coronal Section 2 (FFPE)

Key metrics were:

• Spots detected under tissue - 4,583
• Median UMI counts per spot - 17,129
• Median genes per spot - 5,938

This dataset is licensed under the Creative Commons Attribution license.