10X Genomics obtained fresh frozen Invasive Adenocarcinoma of the large intestine tissue from BioIVT Asterand. The tissue was embedded and cryosectioned as described in Visium Spatial Protocols – Tissue Preparation Guide (Demonstrated Protocol CG000240). Tissue sections of 10µm were placed on Visium Gene Expression slides and fixed and stained following Methanol Fixation, H&E Staining & Imaging for Visium Spatial Protocols (CG000160).
The tissue was AJCC/UICC T4aN0M0, Stage Group IIB.
The H&E image was acquired using a Nikon Eclipse Ti2-E microscope with the following settings:
- Color camera
- 10X objective
- Numerical Aperture: 0.45
- Exposure: 20 ms
The Visium Gene Expression library (T1T2-E8) was prepared as described in the Visium Spatial Reagent Kits User Guide (CG000239 Rev D). Targeted libraries were prepared following the Targeted Gene Expression - Spatial User Guide (CG000377) using the Human Gene Signature Panel (PN-2000285). Targeted sequencing data was processed using Space Ranger.
- Sequencing instrument: Illumina NovaSeq 6000, flow cell HHW57DSXY (lane 4)
- Sequencing depth: 16,795 mean reads per cell
- Sequencing configuration: Paired-end (28 X 90), Dual-Indexed Sequencing. Read 1: 28 cycles (16 bp barcode, 12 bp UMI); i7 index: 10 cycles; i5 index: 10 cycles; Read 2: 90 cycles (transcript).
- Slide: V10A13-206
- Area: C1
Key targeted metrics were:
- Spots detected: 3,138
- Median targeted genes per spot: 351
- Median targeted UMI counts per spot: 635
- Reads mapped confidently to the targeted transcriptome: 88.2%
- Total targeted genes detected: 1,134
To maintain donor anonymity, sequence data for this sample is not currently available.