nuclei rna libraries  (10X Genomics)

Journal: Genome Medicine

Article Title: A chromosomal connectome for psychiatric and metabolic risk variants in adult dopaminergic neurons

doi: 10.1186/s13073-020-0715-x

Figure Lengend Snippet: Phenotypic characterization of Nurr1 + /NeuN + dopaminergic neurons. a (top) Coronal midbrain section including substantia nigra (SN). b Representative FACS plot showing subtypes of sorted nuclei including double positive Nurr1 + /NeuN + (fraction P8). c Midbrain section immunohistochemically stained with anti-Nurr1 antibody, showing Nurr1 immuno-reactive nuclei associated with melanin-positive somata (black arrows) in the SN. d Nurr1 western blot comparing SN versus non-specific midbrain control region. e Dopaminergic marker genes (adopted from single-cell RNA-seq study ) were quantified for expression in the six cell type-specific nuclei fractions collected by FACS from ventral midbrain and anterior cingulate cortex, as indicated. Note significantly higher expression of dopaminergic marker genes ( y -axis) in midbrain Nurr1 + /NeuN + nuclei as compared to other nuclei populations (Wilcoxon test, p < 5 × 10 −2 to 10 −5 ). Note the subtle increase in expression of dopaminergic marker genes in midbrain Nurr1 − /NeuN + nuclei compared to the remaining four cell types, suggesting that this fraction of nuclei represents a more heterogenous admixture of cell types including subset of dopaminergic intermingled with non-dopaminergic neuron nuclei. f Representative genome browser screenshots of nucRNA-seq coverage for dopaminergic marker genes, (top) TYROSINE HYDROXYLASE ( TH ) and (bottom) ENGRAILED1 ( EN1 ) in four midbrain nuclei fractions based on Nurr1 and NeuN immunotagging as indicated and two cortical nuclei fractions based on NeuN immunotagging as indicated: midbrain: (red track) Nurr1 + /NeuN + , (pink track) Nurr1 − /NeuN + ; (orange track) Nurr1 − /NeuN + , (blue track) Nurr1 − /NeuN − ; anterior cingulate cortex (ACC): green track NeuN + and brown track NeuN −

Article Snippet: Nuclei RNA-seq libraries were generated by using SMARTer Stranded RNA-Seq kit following manufacturer’s instructions (Clontech, #634836).

Techniques: Staining, Western Blot, Marker, RNA Sequencing Assay, Expressing

Journal: Nature Communications

Article Title: Mechanisms and function of de novo DNA methylation in placental development reveals an essential role for DNMT3B

doi: 10.1038/s41467-023-36019-9

Figure Lengend Snippet: a The schematic diagram summarises the study design. Embryos were collected at E7.5, after the window of de novo DNA methylation, from Dnmt3a, Dnmt3b , Dnmt3l , Dnmt3a/b DKO and wildtype (WT) controls for bisulphite- and RNA-seq. Phenotyping analysis was done on E12.5 placentas, when all placenta structures and cell types are present. The E12.5 embryo drawings are adapted from Perez-Garcia, V. et al . Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature 555, 463-468 (2018). b The principle component plot shows the distribution of DNA methylation replicates for Dnmt3a , Dnmt3b , Dnmt3l , Dnmt3a/b DKO and WT E7.5 epiblast (Epi) and extra-embryonic ectoderm (ExE), in addition to publicly available data for wildtype E3.5 inner cell mass (ICM) and trophectoderm (TE). DNA methylation was quantitated across autosomes using 100-CpG windows with at least 10 informative CpGs. c The screenshot shows the average DNA methylation for Dnmt3a , Dnmt3b , Dnmt3l , Dnmt3a/b DKO and WT Epi and ExE, using 100-CpG windows with at least 10 informative CpGs.

Article Snippet: Samples were immediately processed to cDNA using the Chromium Controller (10X Genomics), followed by 3’ single-nuclei RNA-seq library preparation (10X Genomics).

Techniques: DNA Methylation Assay, RNA Sequencing Assay

Journal: Nature Communications

Article Title: Mechanisms and function of de novo DNA methylation in placental development reveals an essential role for DNMT3B

doi: 10.1038/s41467-023-36019-9

Figure Lengend Snippet: a Representative haematoxylin and eosin staining of E12.5 placentas from a Dnmt3b KO and littermate WT control (left). The dashed outlined region corresponds to the placental labyrinth zone. The boxplot show the ratio of labyrinth to placental area in Dnmt3b KO ( N = 11) and WT ( N = 10) E12.5 placentas (two-tailed t-test **** p = 0.000002) (right). The boxplot centre line is the median, with box limits showing the upper and lower quartiles and whiskers as 1.5x interquartile range. Individual data points are shown as dots. b Immunofluorescence for MCT1 and MCT4, which stain for syncytiotrophoblast layer I (SynTI) and SynTII that separate maternal and foetal circulation, respectively, of E12.5 placentas from a Dnmt3b KO and littermate WT control. White scale bar = 100 µm. c Representative isolectin BSI-B4 immunohistochemistry of E12.5 placentas from a Dnmt3b KO and littermate WT control, highlighting the matrix surrounding foetal vessels. d t-distributed stochastic neighbour embedding (t-SNE) plots of placental cell types characterised by single-nuclei RNA-sequencing data from E12.5 placentas from Dnmt3b KO and littermate WT control. Tr=Trophoblast; TGCs=trophoblast giant cells. e The scatterplot shows the relative abundance of cell types within the Dnmt3b KO and littermate WT control E12.5 placentas ( p < 0.0001, linear correlation test statistic). f The dot plot shows the difference in expression between Dnmt3b KO and WT for genes upregulated in the Dnmt3a/b DKO ExE ( N = 83). Log2(RPM) was calculated for each gene among cells within the junctional zone and labyrinth from the single nuclei RNA-seq data from E12.5 placenta. Genes that are up- or downregulated in the Dnmt3b KO compared to WT are shown in blue and red, respectively. The proportion of genes up-regulated in Dnmt3b KO was significant in the junctional zone ( p = 0.002) and labyrinth ( p = 0.006) (two-tailed Fisher’s Exact test).

Article Snippet: Samples were immediately processed to cDNA using the Chromium Controller (10X Genomics), followed by 3’ single-nuclei RNA-seq library preparation (10X Genomics).

Techniques: Staining, Two Tailed Test, Immunofluorescence, Immunohistochemistry, RNA Sequencing Assay, Expressing

Journal: Nature Communications

Article Title: Mechanisms and function of de novo DNA methylation in placental development reveals an essential role for DNMT3B

doi: 10.1038/s41467-023-36019-9

Figure Lengend Snippet: a The schematic diagram shows the lineages containing Dnmt3b deletion in the Dnmt3b KO compared to the Dnmt3b cKO, using Sox2-Cre. This schematic is adapted from Perez-Garcia, V. et al . Placentation defects are highly prevalent in embryonic lethal mouse mutants. Nature 555, 463-468 (2018). b Immunofluorescence for MCT1 (SynTI) and MCT4 (SynTII) in E12.5 placentas from a Dnmt3b cKO and littermate WT control. White scale bar = 100 µm. c The scatterplot shows gene expression, evaluated by single-nuclei RNA-sequencing in E12.5 placental cells, in Dnmt3b cKO, using Sox2-Cre, compared to WT littermate control. Cells were pseudo-bulked to generate log2(RPM) values for Dnmt3a/b DKO upregulated DEGs and other genes. d Images show E12.5 embryos from Dnmt3b KO, Dnmt3b cKO and littermate WT controls. Black scale bar = 1 cm. e The bar plot shows the mean observed/expected ratios of KO or cKO embryos at E7.5, E12.5, E18.5 and postnatal day (P)10 in the Dnmt3b KO and cKO crosses, respectively. The number of litters evaluated for the Dnmt3b KO and cKO, respectively, at: E7.5 ( N = 5 and 4), E12.5 ( N = 7 and 5), E18.5 (N = 4 and 7), and P10 (N = 0 and 6). Error bars show standard error of the mean. f E12.5 and E18.5 foetal weights were compared between Dnmt3b cKO ( N = 7 and 7, respectively) and littermate WT (WT and heterozygous) controls ( N = 21 and 21, respectively) from four pregnancies for each time point (two-tailed t-test with Bonferroni correction for multiple comparisons, n.s. = not significant, *** p = 9.1E-10). The boxplot centre line is the median, with box limits showing the upper and lower quartiles and whiskers as 1.5x interquartile range. Individual data points are shown the dots.

Article Snippet: Samples were immediately processed to cDNA using the Chromium Controller (10X Genomics), followed by 3’ single-nuclei RNA-seq library preparation (10X Genomics).

Techniques: Immunofluorescence, Expressing, RNA Sequencing Assay, Two Tailed Test

Journal: Nature neuroscience

Article Title: Disentangling glial diversity in peripheral nerves at single-nuclei resolution

doi: 10.1038/s41593-021-01005-1

Figure Lengend Snippet: Clusters comprising Schwann cells from the ActB-Sun1 and Mpz-Sun1 sciatic nerve atlases were merged to generate a combined Schwann cell atlas. (a) tSNE plot of 10,253 nuclei is shown (middle) with 2 clusters containing cells expressing markers of nmSCs and 4 clusters with expression profiles consistent with mSCs. Markers highlighting subpopulations of nmSCs are shown in top red box ( Prnp, Dbi, B2m and Marcks ) with sCluster5 exhibiting high expression of these 4 genes. Markers highlighting subpopulations of mSCs are shown in bottom blue box (sCluster0: Plp1 ; sCluster1 : Lmna; sCluster3 : Col23a1, Cldn14, Adamtsl1 and Pmp2 ). (b) Comparison of total nerve RNAseq vs Mpz + cell ribosome affinity (Ribo-Tag) RNAseq data analysis of sciatic and vagus nerve reveals 3 sets of Mpz + enriched genes: (i) mSC-enriched genes from sciatic nerve (red), (ii) nmSC-enriched genes from vagus nerve (blue) and (iii) co-enriched genes (green). Each dot represents a gene, and its relative position on the plot indicates the level of enrichment in the ribosome affinity RNAseq ( Mpz + cells) dataset relative to total nerve RNA-seq dataset. (c) Differential expression (DE) analysis comparing vagus and sciatic nerve Mpz + ribosome affinity RNAseq data reveals signatures of myelinating and non-myelinating specific genes (left). Markers of Schwann cell subtypes derived from snRNAseq sciatic nerve atlas display distinct profiles in mSC vs. nmSC-enriched datasets (right). (d) KEGG-based expression module analysis of SC subpopulations. Cldn14/Adamtsl1/Pmp2 + SCs are enriched for NAD and pyruvate metabolic pathways, as well as axon guidance signaling pathway. (e-h) Expression patterns of Cldn14 , Pmp2 , Adamtsl1 and Prx were established using RNA-FISH. Red arrows indicate red signal, green arrows indicate green signal, orange arrows indicate co-localization of red and green signals. Both Pmp2 (e) and Adamtsl1 (f) are expressed in subpopulation of mSCs (marked by Prx). Cldn14 (g) and Adamtsl1 (h) expression overlapped extensively with Pmp2 . Scale bar=50 um. (n=3 biologically independent mice)

Article Snippet: The purified nuclei were used to prepare single nuclei RNA-seq libraries following manufacturer’s instructions (10X Genomics).

Techniques: Expressing, Comparison, RNA sequencing, RNA Sequencing, Quantitative Proteomics, Derivative Assay