GSM5572803: ROI1_2; Homo sapiens; OTHER
To prepare slides for DSP, 4-μm slides from the TMA blocks were deparaffinized and boiled in pressure cooker (Bio SB, Santa Barbara, USA) in Tris-EDTA buffer to expose RNA targets. The slides were digested using GeoMx DSP RNA Slide Prep Kit for FFPE (NanoString, Seattle, USA) to remove RNA-binding proteins and fixed in 16% formaldehyde for 5 min at room temperature. An overnight incubation with GeoMx Panel & Seqcode kit (NanoString) containing probes for 18,676 endogenous targets was then performed in autostainer. Prepared slides were incubated with EpCAM for epithelial cell and 5-HT for EC cell , as well as Syto13 (NanoString) for nuclear identification. After 1 hour incubation at room temperature, slides were washed and stained with fluorescent secondary antibodies at 37℃ for 30 min. After the GeoMx DSP system has completed collection, each GeoMx DSP aspirate in the collection plate contains photocleaved DNA oligos comprised of an RNA analyte identifier, a unique molecular identifier (UMI) barcode, and primer binding sites. When PCR is performed on the aspirates, Illumina adapter sequences and unique dual sample indices are added. Following a pooling purification and quality control process, the final library is ready for sequencing on an Illumina NGS platform using a dualindex workflow.