25 μm formalin-fixed paraffin-embedded (FFPE) curls were sectioned adjacent to sections used for Xenium Prime. Another 25 μm FFPE curls were collected after Xenium Prime sections and pooled together for a total of 50 μm FFPE tissue. Sample preparation was conducted according to Sample Preparation from FFPE Tissue Sections for Chromium Fixed RNA Profiling (CG00632). Approximately 16,500 cells were loaded into GEM wells (targeting 10,000 recovered cells) on a single Chromium X chip. Sequencing libraries were generated following the Chromium Fixed RNA Profiling Reagent Kits for Singleplexed Samples User Guide (CG000691).
Libraries were sequenced on an Illumina NovaSeq 6000 (reagent kit: S4 200 cycles v1.5; loading concentration: 150 nM) with paired-end dual-indexing. The sequencing configuration used:
- 28 cycles Read 1
- 10 cycles i7
- 10 cycles i5
- 90 cycles Read 2
Sequencing libraries were demultiplexed with bcl2fastq (Illumina).
FASTQ files were processed with Cell Ranger v8.0.1 (10x Genomics) using the cellranger multi pipeline and the GRCh38-2020-A reference.
Biomaterials and Pathology
FFPE tissue blocks were purchased from Discovery Life Sciences. Cervical Cancer: III-B (T1b N1 MX)