title: GSM8801540: GC_deep_2G; Homo sapiens; OTHER
library_construction_protocol: The formalin-fixed, paraffin-embedded (FFPE) tissue blocks were cut at 5μm thickness onto APEX treated slides to improve attachment. After incubating at 60°C overnight, deparaffinization, and antigen retrieval was performed on Leica BOND RX autostainer using HIER2 (EDTA-based antigen retrieval solution for 20min) and proteinase K (15min at 37°C, 1μg/ml). RNA probes (NanoString GeoMx WTA Human RNA probes catalog number 121401102) were added for hybridization at 37°C for 16h per protocol. Slides were then washed 2X with stringent wash (50% formamide in 2XSSC for 25min) followed by 2X wash with 2XSSC (2min). Slides were blocked with W buffer for 30min and incubated for 60min at RT with the antibody cocktail containing PanCK-532 RRID:AB_2935724 (1:40), CD45-AF594 RRID:AB_3169472 (1:40), aSMA-AF647(1:200) and Syto 13 (10mM, nuclear stain). Slides were washed with SSC buffer 2X and scanned on the GeoMx DSP machine for selection of ROIs, using FITC/525nm filter to detect nuclei stained with Syto 13 (150msec), Cy3/568 nm channel to detect PanCK (200msec), Texas Red/615 nm to detect CD45 (200msec) and Cy5/666nm to detect aSMA (200msec). Based on tissue morphology, ROIs for EP, SRC, and DGC were selected using polygons, with masking for panCK positive cells. The combined surface area of ROIs was 7699256.57mm2 containing 57880 nuclei. Oligonucleotide barcodes from each ROI were UV-photocleaved, collected into 96-well plates, followed by library preparation and sequencing. Extracted mRNA barcodes were prepared and sequenced on a NextSeq2000 instrument using either a P2 or P3 flow cell kit.