title: GSM8759937: E-C05_S29; Mus musculus; OTHER
library_name: GSM8759937
library_construction_protocol: Sections of paraffin embedded FFPE tissues at 5 μm thickness were placed in a positively charged slide (PathSupply, Wilmington, DE, USA). Sections were mounted on microscope slides and dried.​ The slides were deparaffinized in antigen retrieval solution (Thermo Fisher Scientific) at 100 °C for 20 minutes and treated with 1 µg/mL proteinase K at 37 °C for 15 minutes (Thermo). Mouse transcriptomics was visualized using the GeoMx mouse whole transcriptome atlas (WTA, NanoString, Bruker Spatial Biology, Inc,). An overnight in situ hybridization was performed with a mouse WTA probe and the next day the slides were washed twice at 37 °C for 25 minutes with 50% formamide/2X saline-sodium citrate (SSC) buffer to remove unbound probes. The slides were then incubated with morphological markers. Anti-pancytokeratin (Pan-CK) antibody at 1:200 (Thermo) tagged to Alexa Fluor 532, anti-CD45 antibody at 1:200 (CST) tagged to Alexa Fluor 594, and anti-CD3 antibody at 1:150 (OriGene) tagged to AlexaFluor 647 antibodies were used for immunostaining of slides. Nuclei were stained with Syto13 fluorescent dye (Fluorescence 532, Thermo). Stained slides were loaded onto the GeoMx Digital Spatial Profiler (DSP; NanoString) and scanned. After visual inspection using the GeoMx DSP, geometrical regions of interest (ROI) were selected for oligonucleotide collection. Photocleaved oligonucleotides from each spatially resolved ROI were sequenced with the Illumina NextSeq2000 sequencer (Illumina) and downstream analyses were performed using GeoMx Server (NanoString). After quantifying the probes, the RNA analysis data (DCC files) were transferred to the Nanostring GeoMx platform for detailed QC, normalization, and subsequent analysis. This step facilitated a thorough quality control (QC) assessment using set criteria, the necessary normalization, and analytical procedures. Precise measurement and high-quality data of the RNA samples under study were ensured. As a quality control criterion, the raw read threshold and total number of barcodes were counted by the Illumina sequencer. Only regions with >1000 reads were used for the analysis. Percent aligned reads segments with <80% alignment were flagged for evaluation, assessing potential contamination sources. Sequencing saturation of <50% was flagged. Negative probe count geomean (RNA-NGS) segments of negative probes <10 were flagged, likely to be influenced by slide preparation and wash steps. No template control (NTC) counts of >1000 were analyzed if the DCC file for NTC has low, even count distribution, segments do not have similar deduplicated counts as their NTC, and segments have adequate downstream gene detection. Segments with a cell number or surface area below the threshold were flagged. QC values for % trimmed and % stitched were >90%. Biological QC aimed to remove low-performing probes using one specific probe per gene, focusing on negative probes. A probe was excluded if it was a high or low outlier in >20% of segments. The limit of quantification (LOQ) was defined as two standard deviations above the geometric mean of the negative probes. Segments with low signals were removed retaining segments where 5% of targets were above the expression threshold, set at LOQ below 2. Low-detected genes were removed retaining targets detected in at least 5% of segments as defined by an expression threshold. Technical effects, like segment size and tissue quality, were normalized using third quartile (Q3) normalization. The 75th percentile (Q3) of gene counts was calculated within each segment, and all gene counts were divided by this Q3 value. This step was repeated for each segment, and then all gene counts from all segments were multiplied by the geometric mean of all Q3 counts. Screening genes of interest was performed to eliminate anomalies before consolidating the data into one count per sample and target.