title: GSM8806101: heart, control, Visium replicate 4; Mus musculus; OTHER
library_construction_protocol: Using aseptic, RNA-preserving conditions, control Db/db and T2D db/db mouse hearts were gravity-fed perfusion fixed under deep isoflurane anesthesia (3%) by ~15 seconds of a PBS flush, followed by ~15 seconds of 4% paraformaldehyde (PFA), and finished with 4% PFA with 60 mM KCl until cardiac arrest in diastole. Tissues were fixed for 24 hours in 4% PFA at 4°C and stored in 70% ethanol until paraffin embedding. Paraffin-embedded tissue blocks were sliced in 5 µm sections and transferred to the 6.5 mm x 6.5 mm capture areas on Visium slides according to the 10X Genomics demonstrated protocol (CG000408). Prior to imaging, FFPE tissue mounted on Visium slides were deparaffinized, decrosslinked, and immunofluorescent stained according to the demonstrated protocol from 10X Genomics (CG000410). Multichannel fluorescence images of the whole-heart sections and fiducial frame were captured on a Nikon Ti2-E inverted microscope with a Nikon D-LEDI multiline LED engine and a Hamamatsu ORCA Fusion sCMOS camera. After imaging, sample tissue was prepared using the demonstrated protocol from 10X Genomics (CG000407). Prior to gene expression library construction, quantitative PCR was performed on the eluted probe DNA to determine the appropriate PCR cycle number needed per sample. Following qPCR, probe DNA from the samples were PCR amplified with Dual Index TS Set A (3000511) adapters to make Illumina-compatible libraries for sequencing. Libraries were pooled and sequenced with a target of 25,000 reads/cell using an Illumina NextSeq P2 flow cell.
identifiers.namespace: GEO
identifiers.id: GSM8806101_r1