title: GSM8514156: HCC_TMA_20210616_07_36686_ROI27_Tumor; Homo sapiens; OTHER
library_construction_protocol: We followed published experimental methods (NanoString) with modifications as noted below. Briefly, slides were baked at 60 °C for one hour, deparaffinized with CitriSolv (DECON), rehydrated, antigen-retrieved in 1x Tris-EDTA/pH 9 in a steamer for 15 min at 100 °C, proteinase K (Thermo Fisher Scientific, AM2548) digested at 0.1 ng/ml for 15 min at 37 °C, and postfixed in neutral-buffered formalin for 10 min, hybridized to UV-photocleavable barcode-conjugated RNA in situ hybridization probe set (CTA with 1,779 gene targets and 32 reference genes) overnight at 37 °C, washed to remove off-target probes and then counterstained with morphology markers for 2 h at room temperature. The morphology markers consisted of: 1:50 CD31 (Abcam, ab47063) and 1:50 Arginase-1 (Cell Signaling Technology, 93668S) in blocking buffer W (NanoString). The anti-CD31 antibody was conjugated using the Alexa Fluor 555 Lightning-Link kit (Abcam, ab269820) and the anti-Arginase-1 antibody with the Alexa Fluor 647 Lightning-Link kit (Abcam, ab269823). These two morphology markers allowed distinction between vessel areas of interest (AOIs) and tumor regions of interest (AOIs) within a microscopic region of interest (ROI). 22 Immunofluorescence images, ROI selection, segmentation into marker-specific AOIs and 23 spatially indexed barcode cleavage and collection were performed on a GeoMx DSP instrument 24 (NanoString). Typical exposure times were 400 ms for CD31 and 300 ms for Arg 1. Approximately 25 4 AOIs on average were collected per specimen (details in Table 1) Library preparation was performed according to the manufacturer's instructions and involved PCR amplification to add Illumina adapter sequences and unique dual sample indices. A minimum sequencing depth of 2 150–200 reads per square micron of illumination area was achieved by sequencing all CTA AOIs on a NovaSeq S2 (100 cycles, read 1: 27 nt, read 2: 27 nt, index 1: 8 nt, index 2: 8 nt).