GSM9131715: Cdcp1-KO + AngII/PE, mouse3; Mus musculus; OTHER
FFPE tissue samples sectioned onto standard slides underwent deparaffinization by baking at 60°C for 2h followed by a series of xylene and ethanol treatments. Next, tissue sections were stained for H&E, imaged using the ImageXpress Pico automated cell imaging system (Molecular Devices) at 20x resolution, and then subject to coverslip removal and destaining, using 0.1N HCl for 15 min at 42°C, and decrosslinking using Visium CytAssist kit reagents (10x Genomics). After decrosslinking, tissue sections were hybridized with mouse whole transcriptome probes (10x Genomics) overnight at 50°C in a thermal cycler after which slides were washed, incubated with ligation buffer at 37°C for 1 hr, washed with 2x SSC, stained with eosin, and then inserted into the CytAssist device where the hybridized tissue sections were aligned over the Visium Gene Expression slides (10x Genomics) and processed for probe release for 30 min. In the CytAssist device, a brightfield image of the tissue section was captured. The tissue was processed using manufacture-provided RNase enzyme mix to release the probes from the tissue sections to bind to the capture oligonucleotides located on the Visium Gene Expression slides. Visium Gene Expression slides were incubated to extend the captured probes. Whole transcriptome probes were eluted from the Visium Gene Expression Slide capture areas and subjected to a PCR pre-amplification step, SPRIselect reagent (Beckman Coulter) sample cleanup, and then probe-based library construction using dual index plate TS Set A (10x Genomics). A final SPRIselect sample cleanup was performed, post-library construction quality control assessment completed to verify library 240 bp size by Agilent Bioanalzyer trace peaks, and the resulting libraries were sent for Illumunia sequencing with NextSeq P2, PE100, using paired-end, dual indexed sequencing with the following parameters: Read 1T: 28 cycles, i7 index: 10 cycles, i5 index: 10 cycles, Read 2S: 50 cycles.