GSM9341643: DSP-1001660035646-A-G06; Mus musculus; RNA-Seq
Serial sections of formalin-fixed paraffin-embedded (FFPE) LG tissues harvested from WT (n=3) and TSP-/- (n=4) mice were cut (5 µm thick) and transferred to slides. One set of slides was stained for H&E and another set of slides with consecutive sections were submitted for spatial profiling to the Beth Israel Deaconess Medical Center (BIDMC) Spatial Technologies Unit (STU), MA, USA. The GeoMX Digital Spatial Profiler (DSP) platform was used to generate spatially resolved transcriptomic data, specifically the Whole Transcriptome Atlas (WTA) mouse RNA probe set for Illumina Systems (GMX-RNA-NGS-MsWTA-4) assay. This assay includes RNA probe set for 21,000+ transcripts for mouse protein coding genes plus negative controls designed for Illumina NGS readout with the Seq Code library prep. To visualize tissue structural components and microenvironment, a set of fluorophore-conjugated morphology markers were used. These included Alexa Fluor 532 (green)-conjugated anti-pan-CK (AE1+AE3, Novus, Cat.# NBP2-33200), Alexa Fluor 594 (yellow)-conjugated anti-CD45 (EM-05, Novus, Cat.# NBP1-44763AF594) and Alexa Fluor 647 (red)-conjugated anti-Smooth Muscle Actin (SP171, Abcam, Cat.# ab267537) and nuclear stain SYTO13 (blue). These markers were used to delineate the nuclear, epithelial (acinar and duct), immune and myoepithelial compartments. Immunofluorescence imaging, region of interest (ROI) selection, segmentation into marker-specific areas of interest (AOI) and spatially indexed barcode cleavage and collection were performed on a GeoMx Digital Spatial Profiling instrument (Nanostring). Selected ROIs were exposed to ultraviolet (UV) light to cleave DNA tags in a region-specific manner. The released indexing oligos were then collected using microcapillary aspiration and dispensed into a microplate as per segmentation of each ROI. Four consecutive sections per tissue (total 16 of TSP-1-/- and 12 of WT) were assessed. Approximately, 6-10 ROIs and 11-14 AOIs per tissue sample (total 34 ROIs and 57 AOIs for TSP-1-/- and 21 ROIs and 34 AOIs for WT) were collected. Library preparation was performed as per manufacturer's instructions which included PCR amplification to add Illumina adapter sequences and unique dual sample indices. Sequencing was performed to a minimum of 350M PE reads by pooling all 91 uniquely indexed WTA AOIs and sequencing on Illumina NovaSeq 6000 S4 flowcell in a 2x150bp PE, dual index, 8bp i5 and i7 index length configuration.