GSM8959176: TNBC010|ROI-013|PanCK-; Homo sapiens; OTHER
Tissue sections were prepared following the instruction of GeoMx slide preparation, and the slides were processed using the protocol of RNA slide preparation  for  formalin-fixed paraffin-embedded (FFPE) samples as described in the GeoMx DSP Manual Slide Preparation User Manual (NanoString Technologies, GeoMx-DSP-Manual-Slide-Preparation, MAN-10150). Briefly, deparaffinization and rehydration, antigen retrieval (Invitrogen, IHC Antigen Retrieval Solution, Cat#00-4956-58), and proteinase K digestion (1ug/ml, at 37°C) were performed. Human whole transcriptome atlas (WTA) probes (GeoMx WTA, Mouse RNA Probe for NGS, catalog #121401103) were hybridized in Buffer R (GeoMx RNA Slide Prep Kit PCLN catalog #121300313) and incubated in a hybridization humidity chamber (UVP, HB-1000 Hybridizer) overnight at 37°C. To remove nonspecifically bound probes, slides were washed in 50% Formamide/2x SSC (Thermo Fisher, catalog AM9763, USA) at 37°C (2 washes/25 min each), then slides were blocked and incubated with antibodies diluted with 200 μL Buffer W (GeoMx RNA Slide Prep Kit PCLN catalog 121300313). Then, immunostaining was performed for detection of the region of interest (ROI) . Briefly, Morphology Marker Solution was prepared in the following proportions per slide: 22 μL SYTO (GeoMx Nuclear Stain Morphology Kit, catalog 121300303), 5.5 μL Pan-CK, 5.5 μL CD45 (GeoMx Solid Tumor TME Morphology, Human FFPE RNA Compatible, catalog 121300315), and 181.5 μL Buffer W (GeoMx RNA Slide Prep Kit PCLN catalog 121300313) for a total volume of 220 μL/slide. Slides were incubated in this solution at room temperature for 1 hour followed by 2 consecutive SSC washes, then, slides were loaded into the for ROI selection following GeoMxTM Digital Spatial Profiler Instrument User Manual. After ROI selection, the WTA probe tags were selectively isolated (UV-cleaved) and collected from each of these ROIs, then transferred to individual wells of a collection plate on the DSP. The collections were processed for library preparation following the manual of MANUAL GeoMx® DSP NGS Readout (MAN-10153-06). The quality of library was assessed by the Bioanalyzer DNA 1000 assay (Agilent, catalog 5067-1504). The library was then sequenced on Illumina NextSeq2000. The fastq files were converted to .DCC files using the software of GeoMx-NGS Pipeline; the DCC files were loaded on GeoMx DSP for QC, normalization.  Q3 data was generated and used for all the analysis.