GSM8069250: TC 010; Homo sapiens; OTHER
FFPE tissue sections of 5 µm thickness was prepared according to vendor's protocol with slight modifications. Sections baked at 60°C for 30 min, and incubated sequentially 3x 5 min in xylene, then 2x 5 min each in 100% EtOH, 95% EtOH, and H2O. Antigen retrieved by placing in EDTA at 100°C 20 min, followed by PBS 5 min. Slides then placed in staining jar of 1mg/mL Proteinase K at 37°C 15 min. This was followed by washing in 10% NBF 10 min, 2x washes in NBP stop buffer, and 1x PBS. RNA probe mix was added to tissue slides with a hybridization coverslip at 37°C overnight. Then, slides were washed 2x with SSC buffer and formamide at 37°C, then 2x with SSC buffer. Slides stained for morphology markers SYTO13, PanCK+, VIM, and FAP. Slides were loaded onto NanoString GeoMx DSP for imaging/barcode acquisition, following the manufacturer's protocol. Manual selection of 600-650 µm ROIs and AOIs were performed based on pathology defined areas and morphological markers. Libraries constructed using NanoString NGS readout protocol. Dried aspirates in the collection plate were resuspended with 10 μL of nuclease-free water for 10 min, and 4 μL were transferred to a polymerase chain reaction (PCR) plate. Two μL of 5X PCR Master Mix (NanoString Technologies, Inc., Seattle, WA) and 4 μL of 17 a unique primer mix (NanoString) assigned by their position on the collection plate were added to each well. Contents were mixed by pipetting and the sealed PCR plate was transferred to a thermal cycler for an amplification for 18 cycles. PCR products were pooled in equal amounts. Pooled library was purified twice with a 1.2X magnetic beads-to-sample ratio with KAPA Pure Beads (Roche, Basel, Switzerland), each followed by two 80% ethanol washes, then eluted with Elution Buffer (Nanostring). The quality and quantity of the purified pooled library were assessed with High Sensitivity D1000 ScreenTape System (Agilent, Santa Clara, CA) and Qubit dsDNA High Sensitivity assay (Invitrogen, Waltham, MA), respectively. The library was denatured following Illumina's recommendations and sequenced on NextSeq 550 (Illumina, San Diego, CA) with 27 paired-end cycles, achieving the minimum read depth (30 reads per μm2) or higher determined by the selected AOI's sizes. RNA (amplified oligo nucleotide probes)