NextSeq 500 paired end sequencing; GeoMx digital spatial profiling on biopsies of triple negative breast cancer patients
GeoMx digital spatial profiling on biopsies of triple negative breast cancer patients
Spatial Transcriptomic was performed using the GeoMx DSP® (Nanostring technologies, Seattle, WA, USA) starting from slides of 5μm FFPE diagnostic biopsies. In order to perform spatial selection, slides were stained with  GeoMx Solid Tumor TME Morphology Kit (Nanostring technologies) including  antibodies against CytoKeratin (CK), CD45 and SYTO-13 as nuclear stain. On each slide, the pathologist designed regions of interest (ROIs) representative of the tumor and tumor microenvironment. CK+ regions were classified as tumor, CK- regions were classified as tumor microenvironment. Microenvironment regions were further subdivided in CD45+ (immune-compartment) and CD45- (stroma).  The slides were hybridized with the GeoMx® Cancer Transcriptome Atlas panel according to manufacturer protocol. After AOIs selection and photo-cleavage GeoMx RNA probes were sequenced Hybridized RNA-probes were collected from each of the selected AOIs, and libraries for NGS sequencing were constructed by GeoMx® Seq Code Pack (Nanostring technologies). Libraries were pooled according to AOIs dimension, purified by AMPure XP beads (Beckman Coulter, Brea, Cali-fornia, USA) clean up and finally resuspended in a volume proportional to the number of pooled AOIs. The quality and quantity of library pools were evaluated by Agilent Bioanalyzer.