GSM5416754: Heart.Visium.E13.5.rep2; Mus musculus; RNA-Seq
Embryonic mouse hearts were dissected and saturated in 5%, 10%, 15%, 20% sucrose in PBS, embedded in OCT, and cut to 6 μm sections. Sections were directly placed on capture areas of Visium Spatial Gene Expression Slide (10X Genomics) inside the cryostat chamber. Each of the four capture areas was placed with one Prdm16cKO section and one littermate control section, from one of the 2 pairs of E13.5 or the 2 pairs of E15.5 mouse hearts. After fixation, H&E staining and image capture, sections were incubated with permeabilization enzyme. Optimal tissue permeabilization time was pre-determined to be 8 min for both E13.5 and E15.5 heart sections using Visium Spatial Tissue Optimization Kit (10X Genomics). After permeabilization, mRNA was released from overlying cells and captured by primers on the gene expression spot (~5,000 per capture area). Reverse transcriptase mix was then added to the permeabilized tissue sections and produced spatially barcoded cDNA. Following second strand synthesis and denaturation, the barcoded cDNA was released from capture area and amplified to generate sufficient mass for library construction. After end repair, A-tailing, adaptor ligation and sample index PCR, the library comprised constructs containing the sample index, UMI sequences, spatial barcode sequences, and Illumina standard sequencing primers P5 and P7 at each end.