10x genomics visium pipeline  (10X Genomics)

Journal: Nature Communications

Article Title: Multiplexed transcriptomic analyzes of the plant embryonic hourglass

doi: 10.1038/s41467-024-55803-9

Figure Lengend Snippet: Longitudinal cryosections of Stage 1 embryos embedded in optimal cutting temperature compound (OCT; ( a – b ) two replicates) or in paraffin ( c – d ) six replicates, for use in10x Visium TM spatial transcriptomics analysis of embryo sections. The Toluidine Blue-stained sections ( a , c ) are overlayed with the10x Visium TM spatial map ( b , d ); the various color-coded circles correspond to the capture spots within which spatial cDNA libraries were generated. Scale bars = 100 μm. e Spatial clustering of Visium TM spatial transcriptomic analysis of the Stage 1 embryo. The spatial embryonic origins of each cluster are indicated by the color-coded embryo cartoon; numbers correspond to the cluster designations on the UMAP. f Longitudinal sections of paraffin-embedded Stage 1 embryo for use in LM-RNA-seq. g Enlarged view of a Stage 1 embryo showing regions microdissected for LM-RNA-seq; all LM-RNA-seq analyzes included three replicates. Figure labels: scu, scutellum; uc, upper coleoptile; lc, lower coleoptile. Scale bars =100 μm. h Heatmap illustrating the correlation between the single-cell (sc) clusters ( y -axis) and Visium TM spatial clusters ( x -axis). The significance level is marked by −log 10 p. The p values were calculated based on the cumulative distribution function (CDF) of the one-tailed hypergeometric distribution (for exact p values, please refer to Source Data). i Expression of candidate genes identified in microdissected embryonic organs. Cell clusters exhibiting elevated candidate gene expression of the specific markers are indicated; expression values are log 2 transformed, i.e., log 2 (TPM + 1). sc_cluster, single-cell cluster; V, Visium TM spatial cluster; scu/Scu, scutellum; epi, epidermis; hypo, hypocotyl; ab, abaxial; ad; adaxial; sus, suspensor; col, coleoptile; emb_endo, embryo-endosperm boundary; UC, upper coleoptile; LC; lower coleoptile.

Article Snippet: To further validate the identification of Stage 1 embryo single cell clusters, eight individual Stage 1 embryo sections were processed using the 10X Genomics Visium TM spatial transcriptomics pipeline (Fig. and Supplementary Data ).

Techniques: Staining, Generated, RNA Sequencing Assay, One-tailed Test, Expressing, Transformation Assay

Journal: Nature Communications

Article Title: Multiplexed transcriptomic analyzes of the plant embryonic hourglass

doi: 10.1038/s41467-024-55803-9

Figure Lengend Snippet: a A Venn diagram of embryonic transcriptomics identifies 130 genes co-expressed in initiating embryonic organs. b Normalized expression of these 130 genes at the Proembryo stage (PE), Transition stage (T), Coleoptilar stage (C), Stage 1 scutellum (Scu), Stage 1 upper coleoptile (UC), Stage 1 lower coleoptile (LC), Stage 1 leaf (L), as well as P0 and P1 of the 14 day-after-germination seedlings. The letters above the boxplots represent significance levels (non-overlapping letters indicate significant differences at p < 0.05 using the Tukey-Kramer HSD test (for exact p values, please refer to Source Data). The middle line reflects the median value; the box shows the 25 th and 75 th percentiles and the whiskers reflect 1.5 times the interquartile range. c and d WGCNA ( c ) and hdWGCNA test ( d ) hierarchical cluster tree graph of transcriptomic similarities among tissue-specific co-expression modules, respectively. The height ( y -axis) scales the length of tissue-specific branches, indicating the transcriptomic distances among modules. The hierarchical cluster trees were constructed based on topological overlap dissimilarity, derived from a signed (one-sided) adjacency matrix calculated using the Pearson correlation. In d the significance levels of coexpression module-sc cluster correlations are marked by color-coded circles (significance levels were calculated via −log 10 p; red or blue colors represent relatively strong or weak correlations, respectively); colored branches indicate correlated structures (red branches represent Scutellum-Coleoptile super-cluster; and blue branches represent the SAM&hypocotyl-Leaf super-cluster). The p values (see Supplementary Fig. ) were calculated based on the cumulative distribution function (CDF) of the one-tailed hypergeometric distribution. e Heatmap illustrating expression correlations of the gene set in Maize ( y -axis) and Arabidopsis ( x -axis) during multiple embryonic stages. Heatmap colors are determined by correlation coefficient (r) via Pearson correlation test with significance level p < 0.05 (*) or p < 0.01 (**) (for exact p values, please refer to Source Data). Scu, Stage 1 scutellum; Col, Stage 1 coleoptile; epi: epidermis; hypoc, hypocotyl; M, co-expression modules; sc_cluster, single-cell cluster; PE, Proembryo; T, Transition stage; C, Coleoptilar stage; L: Stage 1 leaf.

Article Snippet: To further validate the identification of Stage 1 embryo single cell clusters, eight individual Stage 1 embryo sections were processed using the 10X Genomics Visium TM spatial transcriptomics pipeline (Fig. and Supplementary Data ).

Techniques: Expressing, Construct, Derivative Assay, One-tailed Test