GSM8546483: neutrophil, DSP-1001660011515-H-G04; Mus musculus; OTHER
Fresh frozen tissues were OCT embedded, and 5um frozen sections were cut with cryostat Fresh frozen tumor sections were mounted onto slides, followed by immunofluorescent staining with specific antibodies targeting protein markers of interest to delineate distinct tissue regions. After staining, high-resolution digital images were acquired to visualize and identify regions of interest (ROIs). ROIs were selected based on morphological and biological relevance. These regions were then subjected to UV light-based spatially resolved photocleavage to release oligonucleotide tags bound to the target mRNA transcripts. The released tags were collected were collected into 96-well plates. Library preparation was performed according to the NanoString GeoMx-NGS Readout Library Prep manual. Briefly, the released tags were dried and reconstituted in 10 µL nuclease free water, and 4 µL was used in a PCR reaction. NanoString barcoded primers were used to amplify the tags and add Illumina adaptor sequences and sample demultiplexing barcodes. PCR products were pooled in equal volumes and purified with two rounds of AMPure XP beads (Beckman Coulter).