Journal: Nature Communications

Article Title: Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

doi: 10.1038/s41467-020-20448-x

Figure Lengend Snippet: a Illustrator and workflow for single-cell RNA sequencing (scRNA-seq) of SAN. b t -distributed stochastic neighbor embedding (t-SNE) analysis identified five clusters including four SAN cell clusters (Clusters 1, 2, 3, 4) and one atrial and ventricular (AV) cardiomyocytes cell cluster (Cluster 5) ( n = 771 biologically independent cells of 25 independent animals). c Heat map shows top differentially expressed genes (DEGs) of each cluster. Cluster 4 was highly expressed cell markers including Hcn4, Hcn1 , and Ednrb, Pde1a and identified as the core cell cluster. d Feature plot of DEGs shows Vsnl1, Unc80 , and Dlgap1 were expressed more specifically than Hcn4 and Hcn1 in Cluster 4. Location of each cluster was marked with the red circle. e Gene Ontology (GO) terms show the biological processes of SAN cell clusters and Cluster 4 was mainly associated with regulation of heart rate and ion transmembrane transport, while Cluster 1, 2, and 3 were all related to electrical activity but had certain difference.

Article Snippet: Primary antibodies included HCN4 (Sigma, SAB5200035, 1:50), Connexin 43 (CST, 3512, 1:50), VSNL1 (Gene Tex, GTX115039, 1:50), Collagen I (Abcam, ab21286, 1:50), DLGAP1 (Affbiotech, AF0308, 1:50), UNC80 (BIOSS, BS-12121R, 1:50), APOLD1 (Novus Biologicals, NBP2-58460, 1:50), RYR3 (Novus Biologicals, NBP2-76962, 1:50), Connexin 40 (Invitrogen, 37-8900, 1:50), cTNT (Abcam, ab8295, 1:50).

Techniques: RNA Sequencing Assay, Activity Assay

Journal: Nature Communications

Article Title: Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

doi: 10.1038/s41467-020-20448-x

Figure Lengend Snippet: a qPCR analysis shows the expression of Hcn4, Vsnl1, Dlgap1, and Unc80 in sinoatrial node (SAN), atrial, and ventricular tissue, respectively ( n = 4 biologically independent animals per group), Dunnett’s multiple comparisons test, data are represented as mean ± s.e.m., adjusted p value was labeled on the top. RA right atrium, LA left atrium, RV right ventricle, LV left ventricle. b – g Immunohistochemical analysis of VSNL1, DLGAP1, and UNC80 in SAN slices at low magnification. Representative images are shown from n = 3 biologically independent samples. b Immunofluorescence images of HCN4 (green), VSNL1 (red), and DAPI (blue). c Immunofluorescence images of HCN4 (green), DLGAP1 (red), and DAPI (blue). d Immunofluorescence images of HCN4 (green), UNC80 (red), and DAPI (blue). b – d The arrows indicate the tissues surrounding the SAN. Scale bar = 250 μm. e – g Localization of the VSNL1, DLGAP1, and UNC80 within SAN at high magnification. e Immunofluorescence images of HCN4 (green), VSNL1 (red), and DAPI (blue). f Immunofluorescence images of HCN4 (green), DLGAP1 (red), and DAPI (blue). g Immunofluorescence images of HCN4 (green), UNC80 (red), and DAPI (blue). Scale bar = 50 μm. e – g Zoom images (the red box regions in the merged image) show the higher magnification. Scale bar = 10 μm.

Article Snippet: Primary antibodies included HCN4 (Sigma, SAB5200035, 1:50), Connexin 43 (CST, 3512, 1:50), VSNL1 (Gene Tex, GTX115039, 1:50), Collagen I (Abcam, ab21286, 1:50), DLGAP1 (Affbiotech, AF0308, 1:50), UNC80 (BIOSS, BS-12121R, 1:50), APOLD1 (Novus Biologicals, NBP2-58460, 1:50), RYR3 (Novus Biologicals, NBP2-76962, 1:50), Connexin 40 (Invitrogen, 37-8900, 1:50), cTNT (Abcam, ab8295, 1:50).

Techniques: Expressing, Labeling, Immunohistochemical staining, Immunofluorescence

Journal: American journal of medical genetics. Part A

Article Title: Phenotypic Evolution of UNC80 Loss of Function

doi: 10.1002/ajmg.a.37929

Figure Lengend Snippet: Identification and characterization of biallelic UNC80 mutations. (A) IGV views of aligned exome sequencing short reads showing paternally inherited NM_032504.1:c.3983-3_3994delinsA. (B) IGV views of aligned exome sequencing short reads showing maternally inherited NM_032504.1:c.2431C>T. Neither mutation has been previously published. (C) Sanger traces for PCR amplification products of NM_032504.1:c.3983-3_3994delinsA. (D) Sanger traces for PCR amplification products of NM_032504.1:c.2431C>T. (E) UNC80 qRT-PCR amplification showing the absence of detectable UNC80 mRNA in cultured skin fibroblasts derived from patient II-3. Target amplification was normalized to that of GAPDH and shown as expression relative to control.

Article Snippet: Quantitative real-time PCR was performed on 100ng of cDNA and UNC80 TaqMan Assay Hs00699496_m1, GAPDH TaqMan Assay Hs03929097_g1, and TaqMan Gene Expression Mastermix (Life Technologies, Carlsbad, CA).

Techniques: Sequencing, Mutagenesis, Amplification, Quantitative RT-PCR, Cell Culture, Derivative Assay, Expressing, Control

Journal: American journal of medical genetics. Part A

Article Title: Phenotypic Evolution of UNC80 Loss of Function

doi: 10.1002/ajmg.a.37929

Figure Lengend Snippet: STRING diagram referenced by Exomiser and showing the proximity of UNC80 in the interactome to NALCN. The edges of this diagram indicate predicted functional links and are colored to indicate supporting evidence for that link (green indicates proteins in the same neighborhood; pink indicates a connection made through experimentation; yellow connections established via text mining for co-occurrence of gene or protein names in abstracts; and light blue indicates relationships supported in databases Biocarta, BioCyc, GO, KEGG, and Reactome). Applying Exomiser to each variant that passed the frequency and segregation filters, we were able to compare the patient’s HPO terms to all human diseases associated with the gene containing the variant in OMIM or Orphanet, to phenotypes associated with orthologs mutated in mice or zebrafish, and lastly to phenotypes associated with nearby genes in the protein-protein association network. For the propositae, Exomiser calculated the phenotype score by considering the similarity of their phenotype to mutation of NACLN (Table I) and the proximity of NACLN to UNC80. The Exomiser variant score is a measure of the predicted pathogenicity and allele frequency of a variant. Exomiser also generated a general score that was calculated from the phenotype and variant scores.

Article Snippet: Quantitative real-time PCR was performed on 100ng of cDNA and UNC80 TaqMan Assay Hs00699496_m1, GAPDH TaqMan Assay Hs03929097_g1, and TaqMan Gene Expression Mastermix (Life Technologies, Carlsbad, CA).

Techniques: Functional Assay, Variant Assay, Mutagenesis, Generated

Journal: American journal of medical genetics. Part A

Article Title: Phenotypic Evolution of UNC80 Loss of Function

doi: 10.1002/ajmg.a.37929

Figure Lengend Snippet: Comparison of features among individuals with UNC80 mutations (table adapted from Perez et al., Shamseldin et al., and Stray-Pedersen et al.)

Article Snippet: Quantitative real-time PCR was performed on 100ng of cDNA and UNC80 TaqMan Assay Hs00699496_m1, GAPDH TaqMan Assay Hs03929097_g1, and TaqMan Gene Expression Mastermix (Life Technologies, Carlsbad, CA).

Techniques: Comparison