GSM7927934: PFA ependymoma, low cell density area, case 38,  sample 2; Homo sapiens; OTHER
Posterior fossa type A (PF-EPN-A) tumor areas with both high and low cell density areas were selected on H&E-stained slides from FFPE tissues. Tissue sections (5 µm) of the corresponding areas were processed according to published protocols [Merritt et al. 2020, PMID: 32393914]. RNA-preserving antigen retrieval was performed, followed by RNA target exposure with proteinase K. In situ hybridization of all targets (n=18,677) on the tissue sections was performed overnight at 37°C, followed by stringent washes to remove off-target probes. Subsequently, antibodies against GFAP (Novus, NBP2-33184DL594, 5 µg/ml), Ki-67 (CST, 9027S, 6 µg/ml), and OLIG2 (Millipore, AB9610, 1:200) were used to visualize target areas as well as DAPI for nuclei counterstains. Three areas of low and high cell density each were selected with the custom polygon region of interest (ROI) tool for each case. These selected areas were illuminated individually via UV light on a GeoMx DSP, resulting in photocleavage of oligonucleotides present within each ROI. Each aspirate contained photocleaved DNA oligos comprising an analyte identifier, a unique molecular identifier (UMI) barcode, and a primer binding site. Illumina adapter sequences and unique dual-sample indices were added when PCR was performed on the aspirates. Sequencing was performed using Illumina's NovaSeq 6000 with the S4 Reagent Kit v1.5 35 cycles flowcell, and paired end reads.