Manual microtome fm2235

The marker genes were further defined with significant gene-gene connectivity in WGNCA analysis (i.e., gene significance greater than 0.4 and weighted correlation index more than 0.4), being differentially expressed genes with a minimal expression level of 1 (UMI index). The electronics in situ hybridization generated by spatial transcriptomics was visualized using Seurat R module of SpatialFeaturePlot^{29 (link)}. The experimental validation of marker genes using RNA in situ hybridization was done following the previous work^{34 (link)}, i.e., the kernel was fixed in 4% paraformaldehyde solution (Sigma) with 0.1% TritonX-100 (Sigma) and 0.1% Tween-20 in PBS (Takara, cat #900) for 16 hours. After dehydration using gradient ethanol and vitrification using xylene, the samples were embedded in paraffin. The kernel sections were cut into 10 μm longitudinally using Leica manual microtome (Leica FM2235). The fragment of the gene coding sequence was cloned and inserted into the pEasy-blunt-zero vector. The antisense and sense RNA probes were transcribed in vitro by T7 and SP6 RNA polymerase according to the instructions for the DIG RNA labeling kit (Roche, catalog number 11175025910). The primers were synthesized at Biosune Biotech (Shanghai, China). The primer sequences used in this study are shown in Table S7.

The marker genes were further defined with significant gene-gene connectivity in WGNCA analysis (i.e., gene significance greater than 0.4 and weighted correlation index more than 0.4), being differentially expressed genes with a minimal expression level of 1 (UMI index). The electronics in situ hybridization generated by spatial transcriptomics was visualized using Seurat R module of SpatialFeaturePlot^{29 (link)}. The experimental validation of marker genes using RNA in situ hybridization was done following the previous work^{34 (link)}, i.e., the kernel was fixed in 4% paraformaldehyde solution (Sigma) with 0.1% TritonX-100 (Sigma) and 0.1% Tween-20 in PBS (Takara, cat #900) for 16 hours. After dehydration using gradient ethanol and vitrification using xylene, the samples were embedded in paraffin. The kernel sections were cut into 10 μm longitudinally using Leica manual microtome (Leica FM2235). The fragment of the gene coding sequence was cloned and inserted into the pEasy-blunt-zero vector. The antisense and sense RNA probes were transcribed in vitro by T7 and SP6 RNA polymerase according to the instructions for the DIG RNA labeling kit (Roche, catalog number 11175025910). The primers were synthesized at Biosune Biotech (Shanghai, China). The primer sequences used in this study are shown in Table S7.