GSM9742510: LEAP_SeqRun_A; Homo sapiens; OTHER
FFPE tissue sections were dried overnight at 37 °C, baked at 60 °C for 1 hour, and processed according to the RNA Slide Preparation Protocol (GeoMx DSP Manual Slide Preparation User Manual; MAN-10150-02). Slides underwent deparaffinisation and rehydration, antigen retrieval in 1x Tris-EDTA for 20 min at approximately 99 °C, RNA target exposure in 0.1 ug/uL Proteinase K for 20 min at 37 °C, post-fix preservation, in situ hybridisation with human Whole Transcriptome Atlas (WTA) RNA probes overnight at 37 °C, and stringent washes in equal parts 4x saline sodium citrate (SSC) buffer and >99.9% formamide. All steps were performed according to MAN-10150-02 unless otherwise specified. Fresh-frozen (FF) tissues were sectioned and submerged in 10% neutral-buffered formalin (NBF) overnight to thaw, baked at 60 °C for 1 hour, and processed according to Appendix II of the RNA Slide Preparation Protocol (MAN-10150-02). This was followed by antigen retrieval in 1x Tris-EDTA for 15 min at approximately 85 °C, RNA target exposure in 0.1 ug/uL Proteinase K for 20 min at 37 °C, in situ hybridisation with human WTA RNA probes overnight at 37 °C, and stringent washes in equal parts 4x SSC and >99.9% formamide. Following stringent washes, all tissues were blocked with Buffer W from the Slide Preparation Kit for 30 min at room temperature and incubated for 1 hour at room temperature with the following antibody solutions sequentially: (1) primary anti-CD45 antibody (2B11 with PD7/26; Novus Biologicals, #NBP2-34528AF594) at 5 ug/mL and primary anti-Ki67 antibody (D2H10; Cell Signaling, #44092) at 6 ug/mL; (2) donkey anti-mouse Alexa Fluor 594 (Jackson ImmunoResearch, #715-586-150) and donkey anti-rabbit Alexa Fluor 647 (Abcam, #ab150075), both at 1:600 dilution; and (3) anti-PanCK (AE-1/AE-3) antibody conjugated with Alexa Fluor 532 (Novus Biologicals, #NBP2-33200AF532) at 2 ug/mL and SYTO 13 Green Fluorescent Nucleic Acid Stain (Thermo Fisher Scientific, #S7575) at 1:1000 dilution. Each incubation was followed by two 5 min washes in 2x SSC buffer to remove unbound reagents. Slides were stored at 4 °C in 2x SSC. Within 21 days of storage, in accordance with NanoString guidelines, regions of interest (ROIs) were selected and segmented into areas of interest (AOIs) containing PanCK+, CD45+, CD45-/PanCK-, or PanCK- cells. GeoMx DSP aspirates were prepared for sequencing as per the GeoMx DSP Readout User Manual. Briefly, aspirates were dried at 60 °C for 1 hour to remove residual liquid, ligated to sequencing primers and spatial barcodes, amplified via PCR, and purified with AMPure beads. Libraries were sequenced on Illumina NextSeq 500/550 or NovaSeq 2000 platforms at a depth of 100 reads per um2.