GSM8751949: DSP-1012550011232-E-A04; Mus musculus; OTHER
Sequencing Saturation is a measure of sequencing depth for a sample [1 – % Unique reads]. Below 50% the counts become less reliable. Sensitivity for low expressing genes is depressed with lower sequencing saturation.  We use Q3 normalization as we find it consistently outperforms other methods like Negative Probe, which can over-normalize small regions and Housekeeper Normalization, which is susceptible to inconsistency in experiments investigating disparate sample types that often have different expression levels. Using a patented DNA barcoding technology, synthetic DNA oligonucleotide barcodes are attached to antibodies for protein detection, and In Situ Probes for mRNA detection via a UV photocleavable linker. A single tissue section is labeled with a cocktail of these probes and 4 fluorescently labeled morphology markers. Regions of interest for molecular profiling are selected based on the fluorescent images, and UV light illuminates each region independent, which releases the barcodes from that region for subsequent counting. Molecular counts for each target in the panel can then be mapped back to distinct regions of interest and comparisons made within and between samples. The workflow is simple with many aspects of it automated in the system.  Staining can be done on an Autostainer, or manually on the bench.  The system first scans the fluorescence, and ROI selection is done right through the software.  Once ROIs are selected, the system goes through and picks from each region one at a time.  As barcodes are released, a microcapillary system sips up the tags, and dispenses them into a 96 well microtiter plate.  Those barcodes can then be read out on our nCounter platform, or an Illumina sequencer.