GSM7102306: Animal C15 Superficial White Matter ROI 125; Macaca nemestrina; OTHER
Unstained 5 µm-thick tissue sections cut from PFA-fixed, paraffin-embedded specimens were mounted on Colorfrost microscope slides In situ hybridizations with the GeoMx Whole Transcriptome Atlas Panel (WTA, 18,676 total targets) were performed in Buffer R Rabbit polyclonal anti-Olig2 antibody was incubated first at 1:100 in Buffer W, followed by Goat anti-rabbit AF647 for visualization. The remaining morphology markers were collectively diluted in Buffer W at the following concentrations: 1:50 RBFOX3 (NeuN), 1:400 GFAP, and STYO 83 for nuclei visualization Each slide was scanned with a 20X objective and default scan parameters on a GeoMx Digitial Spatial Profiler ROI placement: Geometric 500 µm diameter circle ROIs were placed in the subcortex (Superficial WM; n=3/section), WM tracts (Deep WM; n=3/section), and cortical layers IV-VI (GM; n=3/section). One maximum area (660 µm X 785 µm) ROI was placed at the GM/WM interface on each section ALOI segmentation: Segmentation mask was applied to the GM/WM interface maximum area to select Olig2+, GFAP+, and NeuN+ areas of interest (AOI). Sequencing libraries were generated by PCR from the photo-released indexing oligos and ROI-specific Illumina adapter sequences and unique i5 and i7 sample indices were added Total target counts per DSP collection plate for sequencing were calculated from the total samples areas (µm2). For WTA libraries, the target sequencing depth was 100 counts/µm2. Pooled libraries were sequenced at 2×27 base pairs and with the dual-indexing workflow on an Illumina NovaSeq