GSM4861202: Visium-A1; Mus musculus; OTHER
Tissue preparation was performed according to the Visium Tissue Preparation Guide (CG000240 Rev A, 10X Genomics). Briefly, the trunk of 3 stage-matched E9.5 Pax2-GFP mouse embryos was dissected  in cold DMEM containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, embedded in Optimal Cutting Temperature (OCT) VWR clear frozen section compound and cryosectioned at 10 μm. Some sections were placed in pre-chilled DNA LoBind microcentrifuge tubes 1.5 ml (Eppendorf) for total RNA extraction and assessment of  average RNA integrity (RIN) quality score. RNA quality of the samples was good, RIN score=9.5, as defined using 2200 TapeStation (Agilent) with High Sensitivity RNA ScreenTape Assay (Agilent). Other sections were placed in pre-chilled Visium Tissue Optimization Slides (10X Genomics) or Visium Spatial Gene Expression Slide (10X Genomics) for tissue optimization and gene expression experiments, respectively, and stored  at -80 °C for less than a week. Tissue slides were processed following Visium guidelines (Visium Spatial Tissue Optimization User Guide (CG000238 Rev A, 10X Genomics) or Visium Spatial Gene Expression User Guide (CG000239 Rev A, 10X Genomics). Tissue sections on the gene expression slide were permeabilized for 12 minutes, which was pre-determined as the optimal time established after the Tissue Optimization experiments. Brightfield histology images for Tissue Optimization were taken using a 10x objective on an EVOS FL Auto imaging system (ThermoFisher Scientific). Fluorescent images were taken with a TRITC filter using a 10X objective and 300 ms exposure time. For Tissue Expression, brightfield images were taken using a 20X objective on a Nikon Eclipse Ti2 microscope. Raw images were stitched together using NIS-Elements AR 5.11.00 (Nikon) and exported as .tiff files with low- and high-resolution settings. Library preparation was performed according to the Visium Spatial Gene Expression User Guide (CG000239 Rev A, 10X Genomics). Libraries were loaded and sequenced on a NovaSeq 6000 System (Illumina) as paired-end-dual-indexed with NovaSeq SP PE150 Reagent Kit (Illumina), at  a sequencing depth of approximately 25M read-pairs per capture area.