Species and cell-type properties of classically defined human and rodent neurons and glia
- Howard Hughes Medical Institute, The Rockefeller University, United States
- University of Miami, United States
Figures
Figure 1 with 2 supplements
Generation of gene expression profiles for distinct cell types from the cerebella of wild-type mice.
(A) Immunofluorescence staining of five distinct cell types in the cerebellum. Antibodies used to label each cell type: NeuN for granule cells, ITPR1 for Purkinje cells, SORCS3 for basket cells, …
Figure 1—figure supplement 1
Nuclear RNA profiles can specify cell-type identity, related to Figure 1.
(A) EGFP-L10a expression from five bacTRAP animals. Neurod1, Pcp2, and Sept4 drive expression in granule cells, Purkinje cells, and Bergmann glia of the cerebellum. Colgalt2 and Ntsr1 drive …
Figure 1—figure supplement 2
Overview of nuclei labeling and sorting strategy and comparison to single nuclei sequencing, related to Figure 1.
(A) Schematic showing the strategy for cell-type specific nuclei purification and gene expression profiling. Starting from whole tissue containing heterogeneous cell populations intermingled …
Figure 2 with 1 supplement
Generation of gene expression profiles for distinct cell types from rat and human cerebella.
(A) Fluorescence activated sorting of stained nuclei from six cell types in the rat cerebellum. Antibodies are indicated on the x and y axis. When a cell type can be isolated from more than one …
Figure 2—figure supplement 1
Analysis of cell type specific gene expression profiles generated from rat and human cerebella identifies known mouse marker genes for each cell type, related to Figure 2.
(A) Heatmap showing the 20 most specific genes for each rat cell type as identified by the Specificity Index algorithm. Rows: cell-type specific samples; columns: genes. Color represents the z-score …
Figure 3 with 3 supplements
Comparative analysis of gene expression across species reveals cell-type and species specific differences.
(A) Heatmap showing normalized expression of the 250 most variable genes across all samples. Hierarchical clustering is performed using these genes and reveals that samples cluster primarily by cell …
Figure 3—figure supplement 1
Comparative analysis of gene expression in specific cell types of mouse, rat, and human, related to Figure 3.
(A) Schematic for defining ortholog annotation for mouse, rat, and human genes. Ortholog annotation across species were downloaded from ENSEMBL, filtered to include only high confidence pairs, 1:1 …
Figure 3—figure supplement 2
Detailed comparative analysis of gene expression in five cerebellar cell types in mouse and human, related to Figure 3.
(A) Schematic for defining ortholog annotations for mouse and human genes. Ortholog annotation across species were downloaded from ENSEMBL, filtered to include only high confidence pairs, 1:1 …
Figure 3—figure supplement 3
Expression of species-enriched genes in published cerebellar single nuclei/cell RNA-seq data, related to Figure 3.
For all figures, human single nuclei RNA-seq data is from (Lake et al., 2018) and mouse single cell RNA-seq data is from (Saunders et al., 2018). Author designations for cell types are used except …
Figure 4 with 2 supplements
Epigenetic and immunofluorescence validation of gene expression differences between mouse and human cerebellar granule cells.
(A) Browser views showing a homologous region of approximately 150 kb from chr14 in human and chr12 in mouse. Minimum and maximum data range values are indicated for each track. Genes located in …
Figure 4—figure supplement 1
Analysis of cell-type specific ATAC peaks and examples of mouse- and human-enriched genes in granule cells, related to Figure 4.
(A-B) Browser view and heatmap showing chromatin accessibility by ATAC-seq (dark green) and nuclear gene expression levels by RNA-seq (green) from granule cells from human (red) and mouse (blue). (A)…
Figure 4—figure supplement 2
Relationship between chromatin accessibility and gene expression in cerebellar granule and basket neurons, related to Figure 4.
(A) Stacked bar plot showing the proportion of peaks identified from ATAC-seq DNA accessibility assay from human and mouse cerebellar granule and basket neurons that map to various genomic regions. …
Figure 5
Profiling of three cell types in 16 human cerebellar samples.
(A) Fluorescence activated nuclear sorting of three cell types from the cerebella of 14 individuals. The percentage of each cell type in different individuals is shown. Each sample is identified by …
Figure 6 with 1 supplement
Clinical factors impact gene expression in a cell-type specific manner.
(A,B) GYG2P1: a basket-specific, male-specific gene. (A) quantification of GYG2P1 gene expression in granule, basket, and glial nuclei for female and male samples. (B) browser view showing …
Figure 6—figure supplement 1
Analysis of clinical factors that contribute to gene expression variability across individuals, related to Figure 6.
(A) Table showing the number of differentially expressed genes (adjusted p<0.01, baseMean >50) for all cell types or each cell type individually when samples are stratified by clinical factors. For …
Figure 7 with 1 supplement
Additional sources of interindividual variability in gene expression.
(A, B) Principal component analysis of samples for each cell type identifies interindividual gene expression variability. (A) Plot showing the proportion variance explained by each of the first …
Figure 7—figure supplement 1
Additional analyses of interindividual variability in gene expression, related to Figure 7.
(A) For each cell type, plots showing loadings (left) and scores (right) for PC1 and PC3 (top row) or PC1 and PC4 (bottom row). For loadings, the five genes with the highest absolute values from the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain (M. musculus) | NeuroD1 EGFP-L10a | (Doyle et al., 2008) | RRID:IMSR_JAX:030262 | |
| Strain (M. musculus) | Pcp2 EGFP-L10a | (Doyle et al., 2008) | RRID:IMSR_JAX:030267 | |
| Strain (M. musculus) | Sept4 EGFP-L10a | (Doyle et al., 2008) | RRID:IMSR_JAX:030271 | |
| Strain (M. musculus) | Glt25d2 EGFP-L10a | (Doyle et al., 2008) | RRID:IMSR_JAX:030257 | |
| Strain (M. musculus) | Ntsr1 EGFP-L10a | (Doyle et al., 2008) | RRID:IMSR_JAX:030264 | |
| Strain (M. musculus) | Wild type | Jackson labs | RRID:IMSR_JAX:000664 | |
| Antibody | anti-NeuN | Abcam | RRID:AB_2532109 | Rabbit monoclonal; 1:250 for nuclei staining; 1:250 for IF |
| Antibody | anti-NeuN | Millipore | RRID:AB_2298772 | Mouse monoclonal; 1:250 for nuclei staining; 1:250 for IF |
| Antibody | anti-Itpr1 | Abcam | ab190239 | Mouse monoclonal; 1:500 for nuclei staining; 1:500 for IF |
| Antibody | anti-Sorcs3 | Thermo | RRID:AB_2606387 | Goat polyclonal; 1:250 for nuclei staining; 1:250 for IF |
| Antibody | anti-EAAT1 | Abcam | RRID:AB_304334 | Rabbit polyclonal; 1:250 for nuclei staining |
| Antibody | anti-Olig2 | R and D | RRID:AB_2157554 | Goat polyclonal; 1:250 for nuclei staining |
| Antibody | anti-GFAP | Abcam | RRID:AB_880202 | Goat polyclonal; 1:500 for IF |
| Antibody | anti-Mog | Thermo | RRID:AB_2607363 | Goat polyclonal; 1:500 for IF |
| Antibody | anti-Mag | Cell Signaling | RRID:AB_2665480 | Rabbit polyclonal; 1:500 for IF |
| Antibody | anti-Pde1a | Acris | TA311317 | Goat polyclonal; 1:200 for IF |
| Antibody | anti-Pde1c | Santa Cruz | RRID:AB_11149544 | Rabbit polyclonal; 1:100 for IF |
| Sequence-based reagent | raw and processed sequencing data | this paper | GSE101918 | Details about all samples in this superseries are in Supplementary file 1 |
| Sequence-based reagent | raw and processed sequencing data | (Mo et al., 2015) | GSE63137 | Details about all samples in this superseries are in Supplementary file 1 |
| Sequence-based reagent | raw and processed sequencing data | (Habib et al., 2016) | GSE85721 | Details about all samples in this superseries are in Supplementary file 1 |
| Sequence-based reagent | raw and processed sequencing data | (Lake et al., 2018) | GSE97930 | Details about all samples in this superseries are in Supplementary file 1 |
| Sequence-based reagent | raw and processed sequencing data | (Saunders et al., 2018) | GSE116470/dropvis.org | Details about all samples in this superseries are in Supplementary file 1 |
| Commercial assay or kit | AllPrep FFPE | Qiagen | 80234 | |
| Commercial assay or kit | Rneasy Micro | Qiagen | 74004 | |
| Commercial assay or kit | MinElute Reaction Cleanup | Qiagen | 28206 | |
| Commercial assay or kit | Ovation RNAseq V2 | Nugen | 7102–32 | |
| Commercial assay or kit | Ultra II DNA Lirary Prep Kit for Illumina | NEB | E7645L | |
| Commercial assay or kit | Multiplex Adapters for Illumina | NEB | E7335L, E7500L | |
| Commercial assay or kit | Bioanalyzer Pico chips | Agilent | 5067–1513 | |
| Commercial assay or kit | TapeStation D1000 ScreenTape | Agilent | 5067–5583 | |
| Commercial assay or kit | TapeStation High Sensitivity D1000 ScreenTape | Agilent | 5067–5585 | |
| Software, algorithm | source code | (Xu, 2018) | https://github.com/xu-xiao/non_transgenic_cell_type_profiling | R scripts for analysis and generating figures; parameters for running command line tools. |
Additional files
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Supplementary file 1
Summary of all RNA-seq datasets, including information about animals, sorts, and quality control metrics related to methods.
Also includes information on published RNA-seq datasets used in this manuscript.
- https://doi.org/10.7554/eLife.37551.019
-
Supplementary file 2
Clinical information for all human tissue donors, related to methods.
- https://doi.org/10.7554/eLife.37551.020
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Supplementary file 3
Specificity index calculations for mouse, rat, and human cell types using either species-specific annotations or with mouse-rat-human orthologous gene annotations.
SIs for mouse and rat samples with orthologous gene annotations have been calculated using either all cell types (all) or without Purkinje samples.
- https://doi.org/10.7554/eLife.37551.021
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Supplementary file 4
Differential expression analysis results for mouse and human-enriched genes.
Also mouse and human IDs for genes that are unchanged in gene expression between the two species.
- https://doi.org/10.7554/eLife.37551.022
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Supplementary file 5
Annotation of ATAC peaks.
All: all MACS2 called peaks; DA: differentially accessible peaks between granule and basket neurons.
- https://doi.org/10.7554/eLife.37551.023
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Supplementary file 6
Motif analysis of various ATAC-seq defined regions.
DA: regions that are differentially accessible between granule (gran) and basket (bsk) neurons in mouse (m) or human (h); HE: regions defined by peaks located in the promoter (p) or gene body (gb) of human (h) enriched genes for granule (gran) or basket (bsk) neurons; ME: regions defined by peaks located in the promoter (p) or gene body of mouse (m) enriched genes for granule (gran) or basket (bsk) neurons.
- https://doi.org/10.7554/eLife.37551.024
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Supplementary file 7
Differentially accessible regions between human granule and basket neurons that contain single nucleotide polymorphisms (SNPs) associated with human disease.
The column Multiple specifies whether a SNP has been linked to a specific disease/trait in at least two publications.
- https://doi.org/10.7554/eLife.37551.025
-
Supplementary file 8
Differential expression analysis results for the influence of clinical factors on gene expression in human samples.
- https://doi.org/10.7554/eLife.37551.026
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Supplementary file 9
Full results from all gene ontology (GO) analyses performed in the paper.
- https://doi.org/10.7554/eLife.37551.027
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Transparent reporting form
- https://doi.org/10.7554/eLife.37551.028
