Defining cellular and molecular identities within the kidney is necessary to understand its organization and function in health and disease. Here we demonstrate a reproducible method with minimal artifacts for single-nucleus Droplet-based RNA sequencing (snDrop-Seq) that we use to resolve thirty distinct cell populations in human adult kidney. We define molecular transition states along more than ten nephron segments spanning two major kidney regions. We further delineate cell type-specific expression of genes associated with chronic kidney disease, diabetes and hypertension, providing insight into possible targeted therapies. This includes expression of a hypertension-associated mechano-sensory ion channel in mesangial cells, and identification of proximal tubule cell populations defined by pathogenic expression signatures. Our fully optimized, quality-controlled transcriptomic profiling pipeline constitutes a tool for the generation of healthy and diseased molecular atlases applicable to clinical samples. Overall design: Single-nucleus (sn)Drop-seq was used to generate RNA expression estimates across two kidney regions (cortex and medulla), 15 different individuals, 7 different tissue processing methods, and from tissues acquired from two different institutions (Washington University and University of Michigan through KPMP consortium). From the resulting ~18,000 sequenced nuclei passing QC filtering (>400 <5000 non-MT genes detected, >50 post-QC nuclei per library, >30 nuclei per cluster), we identified 30 different cell populations (see supplementary file UCSD-WU_Single_Nuclei_Cluster_Annotations.csv).
A single-nucleus RNA-sequencing pipeline to decipher the molecular anatomy and pathophysiology of human kidneys.
Sex, Specimen part, Subject
View SamplesDCA (3,5-Dichloroanthranilic acid) is a newly identified synthetic defense elicitor. To perform a comparative analysis of defense responses triggered by DCA and the structurally related defense inducer INA (2,6-Dichloroisonicotinic acid) Affymetrix chip experiments were performed with Arabidopsis thaliana seedlings treated with one of these two compounds.
The synthetic elicitor 3,5-dichloroanthranilic acid induces NPR1-dependent and NPR1-independent mechanisms of disease resistance in Arabidopsis.
No sample metadata fields
View SamplesWe characterzised global changes in gene expresseion between 8 cell embryos and blastocysts to identify potential genes required for blastocyst formation.
Transcription factor AP-2γ is a core regulator of tight junction biogenesis and cavity formation during mouse early embryogenesis.
Specimen part
View SamplesIn germ cells, Piwi proteins interact with a specific class of small non-coding RNAs, piwi-interacting RNAs (piRNAs). Together, these form a pathway that represses transposable elements, thus safeguarding germ cell genomes. While basic models describe the operation of piRNA pathways, neither the protein compositions of Piwi complexes, the critical protein-protein interactions that drive small RNA production and target recognition, or the precise molecular consequences of conserved localization to germline structures, call nuage, is well understood. We purified the three murine Piwi family proteins, Mili, Miwi, and Miwi2, from mouse germ cells and characterized their interacting protein partners. Piwi proteins were found in complex with Prmt5/Wdr77, an enzyme that di-methylates arginine residues. By immunoprecipitation with specific antibodies and by mass spectrometry, we found that Piwi proteins are arginine methylated at conserved positions in their amino termini. These modifications are essential to direct complex formation with specific Tudor-domain proteins, whose interactions with Piwis can be required for localization of RNP complexes in cytoplasmic nuage, proper piRNA expression, and transposon silencing. Considered together, our findings indicate that arginine methylation drives the assembly of multi-protein machines whose integrity and specific sub-cellular localization is necessary for efficient function of the piRNA pathway. Keywords: gene regulation study Overall design: Total small RNA in embryonic and post-birth mouse testes of tdrd1 and tdrd6 mutants
RNF17 blocks promiscuous activity of PIWI proteins in mouse testes.
No sample metadata fields
View SamplesGenome-wide gene expression was obtained in three auditory brainstem nuclei (defined below), at two different ages in mice, postnatal day (P)3 and P14. The primary aim was to identify genes which are differentially expressed between the medial nucleus of the trapezoid body (MNTB) and the superior olive (LSO), at both age groups.
BMP signaling specifies the development of a large and fast CNS synapse.
Sex, Specimen part
View SamplesThe function of Structural maintenance of chromosome flexible domain containing 1 (Smchd1) was examined during mouse preimplantation development using an siRNA knockdown approach. Transient SMCHD1 deficiency during the period between fertilization and morula/early blastocyst stage compromised embryo viability and resulted in reduced cell number, reduced embryo diameter, and reduced nuclear volumes at the morula stage. RNAseq analysis of Smchd1 knockdown morulae revealed aberrant increases in expression of mRNAs related to the trophoblast lineage, indicating SMCHD1 inhibits trophoblast lineage gene expression and promotes inner cell mass formation. siRNA knockdown also reduced expression of cell proliferation genes, including S-phase kinase-associated protein 2 (Skp2). Smchd1 expression was elevated in Caudal type homeobox transcription factor 2 (Cdx2)-/- blastocysts, indicating enriched expression, and further indicating a role in inner cell mass development. These results indicate that Smchd1 plays dual roles in the preimplantation embryo, promoting a lineage-appropriate pattern of gene expression supporting inner cell mass formation, whilst controlling lineage formation and gene expression in the trophectoderm. Overall design: Effects of SMCHD1 siRNA knockdown were tested in mouse embryos
Novel key roles for structural maintenance of chromosome flexible domain containing 1 (Smchd1) during preimplantation mouse development.
Treatment, Subject
View SamplesshRNAs were assessed for off-target effects by comparing the gene expression profiles of cells that they had been infected into. shRNAs designed with the shERWOOD algorithm and house in the ultramir microRNA scafold were found to have very little off targeting. Overall design: Purpose: A major detriment to RNAi is off-targeting. We wished to assess the level of off targeting of microRNA (ultramiR) housed shERWOOD shRNAs as compared to similar shRNAs in the TRC collection. Methods: 5 shRNAs targeting each of two genes were infected into the 4T1 cell line. For each gene one shRNA was selected from the TRC collection and one based on the shERWOOD algorithm. For each gene, the exrpession profiles of the corresponding shRNA infected cells were compared using RNAseq. Conclusions: Highly similar profiles were observed between shERWOOD selected shRNAs. TRC shRNAs produced profiles indicative of off-targeting.
A computational algorithm to predict shRNA potency.
No sample metadata fields
View SamplesThe transcriptional profile 23 cell lines derived from single clones of the 4T1 cell lines were assessed with RNAseq. The two clones with a strong propensity to intravasate were found to have 12 genes in common that were overexperessed relative to the other 21 clones. Overall design: Clone RNAseq 1) 23 clonal lines were established using single cell FACs sorting from the 4T1 mammary cancer cell line. 2) After establishing the lines the clones were assesed (in a pooled setting) for their capacity to intravasate the vascular system. 3) Transcriptional profiling was carried out using RNAseq. 4) Two clones were found to be strong intravasators and these were compared to the other clones to identify genes that were overexpressed (as compared to at least half of the other clones in both lines).
A model of breast cancer heterogeneity reveals vascular mimicry as a driver of metastasis.
No sample metadata fields
View SamplesWe used full genome microarrays to profile the full lifetime of the mouse placenta from embryonic day 8.5 (e8.5), at the time of chorioallantoic fusion, until postnatal day 0 (P0).
Genomic evolution of the placenta using co-option and duplication and divergence.
Specimen part
View SamplesWe used full genome microarrays to profile the full lifetime of the mouse placenta from embryonic day 8.5 (e8.5), at the time of chorioallantoic fusion, until postnatal day 0 (P0). For these samples, at each stage the fetal placenta and maternal decidual tissues were dissected and profiled separately (See series 1).
Genomic evolution of the placenta using co-option and duplication and divergence.
Specimen part
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