GSM8516219: LC01_Pre_S1; Homo sapiens; OTHER
For spatial transcriptomic profiling, tissue sections from multiple patients were placed on the same slide (three slides in total), and 4 μm sections of the tissue microarray (TMA) were used for histological analysis following hematoxylin and eosin staining. Serial sections were stained simultaneously with fluorescently labeled antibodies to detect pan-cytokeratin (NanoString, AE1/AE3, cat. no. 121301310), and DNA-binding dye (ThermoFisher, Syto13, cat. no. S7575) was then applied to the slides. Using the instrument's web interface, ROIs up to 780 × 660 μm were selected and outlined by two experienced pathologists (JK and YKJ). Library preparation was performed according to manufacturer instruction (Nanostring DSP-Genomics Library Preparation Protocol, Nanostring Technologies, Seatle, WA) (Merritt et al., 2020). Photocleaved oligonucleotides from each ROI were PCR amplified using Illumina's i5/i7 dual-indexing system to preserve ROI identity. PCR products were pooled and purified with two rounds of AMPure XP beads (Beckman Coulter, Brea, CA). Bioanalyzer High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA) was used to measure library concentration and purity. Paired-end (2 75 bp reads) sequencing was performed on an Illumina NextSeq instrument. Reads were then aligned to analyte barcodes with Bowtie