Molecular pathway analysis towards understanding tissue vulnerability in spinocerebellar ataxia type 1
- Yale School of Medicine, Unites States
Figures
Figure 1
Schematic illustrating the cross-tissue and cross-model comparisons conducted to identify common and unique molecular alterations across SCA1 affected tissues.
Transcriptomics data from Atxn1154Q/2Q KI inferior olive and cerebellum were first analyzed individually (1-2) before comparing the two tissues (3). Using the ATXN1-82Q Tg inferior olive, comparison …
Figure 2 with 4 supplements
Defense Response-related genes are significantly enriched in the 12 week old Atxn1154Q/2Q KI inferior olive.
(A) PolyQ-expanded Atxn1 is expressed in the inferior olive of Atxn1154Q/2Q KI mice (n = 2 animals per genotype). * marks Atxn1-2Q and ** marks Atxn1-154Q. (B) Illustration of the brain region …
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Figure 2—source data 1
5 week old Atxn1154Q/2Q KI inferior olive functional annotation clustering enrichment analysis.
- https://doi.org/10.7554/eLife.39981.008
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Figure 2—source data 2
Functional annotation enrichment analysis for the 12 week old Atxn1154Q/2Q KI inferior olive.
- https://doi.org/10.7554/eLife.39981.009
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Figure 2—source data 3
Upstream regulators for differentially regulated 12 week old Atxn1154Q/2Q KI inferior olive genes.
- https://doi.org/10.7554/eLife.39981.010
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Figure 2—source data 4
5 week old Atxn1154Q/2Q KI inferior olive enriched pathways for cross time-point overlap with EnrichmentMap (enrichment FDR p-value < 0.05).
- https://doi.org/10.7554/eLife.39981.011
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Figure 2—source data 5
12 week old Atxn1154Q/2Q KI Inferior Olive enriched patwhays for cross-time point overlap with EnrichmentMap.
- https://doi.org/10.7554/eLife.39981.012
Figure 2—figure supplement 1
Response to Organic Substance and Hormone Metabolic Process genes are differentially regulated in the 5 week old Atxn1154Q/2Q KI inferior olive.
(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in Atxn1154Q/2Q KI inferior olive at 5 weeks of age (FDR p-value < 0.05; n = 3 males per genotype). (C) …
Figure 2—figure supplement 2
Features of the Defense Response, including Interferon Signaling and IRF Activation, are predicted to have increased activity in the 12 week old Atxn1154Q/2Q KI inferior olive.
(A) Biological pathway enrichment using Qiagen’s IPA in the 12 week old Atxn1154Q/2Q KI inferior olive differentially expressed genes. X-axis marks the enrichment score using a Benjamini-Hochberg …
Figure 2—figure supplement 3
Subset of genes associated with the enriched biological pathway Neurological System Process are down-regulated in the 12 week old Atxn1154Q/2Q KI inferior olive.
(A) Down-regulated genes within the Neurological System Process pathway (** FDR p-value < 0.01; n = 3 males per genotype used in RNA-seq analysis). (B) Validation of Calb1 down-regulation using …
Figure 2—figure supplement 4
Divergent gene expression changes and minimal biological pathway overlap in the temporal comparison of the Atxn1154Q/2Q KI inferior olive.
(A) Schematic of the time-point comparison in Atxn1154Q/2Q KI inferior olive between 5 and 12 weeks. (B) Total number of differentially regulated genes in the Atxn1154Q/2Q KI inferior olive relative …
Figure 3 with 4 supplements
Chemical Synaptic Transmission genes, including GABAergic and glutamatergic genes, are differentially regulated in the 12 week old Atxn1154Q/2Q KI cerebellum.
(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in the 12 week old Atxn1154Q/2Q KI cerebellum (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). (C)…
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Figure 3—source data 1
5 week old Atxn1154Q/2Q KI cerebellum functional annotation clustering (differentially expressed genes FDR p-value < 0.05).
- https://doi.org/10.7554/eLife.39981.019
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Figure 3—source data 2
12 week old Atxn1154Q/2Q KI cerebellum functional annotation enrichment analysis.
- https://doi.org/10.7554/eLife.39981.020
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Figure 3—source data 3
5 week old Atxn1154Q/2Q KI cerebellum functional annotation enrichment analysis (using nominal p-value < 0.01 for genes).
- https://doi.org/10.7554/eLife.39981.021
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Figure 3—source data 4
Atxn1154Q/2Q KI Cerebellum 5 weeks enrichment chart edited for EnrichmentMap (nominal p-value < 0.01).
- https://doi.org/10.7554/eLife.39981.022
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Figure 3—source data 5
12 week old Atxn1154Q/2Q KI cerebellum enrichment chart edited for EnrichmentMap (FDR p-value < 0.05).
- https://doi.org/10.7554/eLife.39981.023
Figure 3—figure supplement 1
Few genes and pathways are significantly enriched in the 5 week Atxn1154Q/2Q KI cerebellum.
(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in Atxn1154Q/2Q KI cerebellum at 5 weeks of age (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). …
Figure 3—figure supplement 2
Consistent gene expression changes in the Atxn1154Q/2Q KI cerebellum across the 5 and 12 week time-points.
(A) Schematic of the time-point comparison in Atxn1154Q/2Q KI cerebellum. (B) Total number of differentially regulated genes in the Atxn1154Q/2Q KI cerebellum that are commonly altered in both the 5 …
Figure 3—figure supplement 3
Nominally significant differentially regulated genes in the 5 week old Atxn1154Q/2Q KI cerebellum overlapped with previously published microarray datasets and revealed significantly enriched pathways.
(A) Subset of genes with a nominal p-value < 0.01 that overlapped with a previously published dataset (* nominal p-value < 0.01 but FDR p-value > 0.05; n = 3 males per genotype used in RNA-seq). (B) …
Figure 3—figure supplement 4
Consistent gene expression changes and pathway enrichment in the 5 week and 12 week Atxn1154Q/2Q KI cerebellum with an expanded 5 week p-value cutoff.
(A) Schematic of the time-point comparison in Atxn1154Q/2Q KI cerebellum. (B) Total number of differentially regulated genes in the Atxn1154Q/2Q KI cerebellum relative to appropriate WT controls …
Figure 4 with 1 supplement
Cross-tissue comparison of pathway enrichment reveals common and unique features of the 12 week old Atxn1154Q/2Q KI inferior olive and cerebellum.
(A) Schematic of the cross-tissue comparison conducted. (B) Clustering of GO terms from the 12 week old inferior olive and cerebellum differentially expressed genes lists. In this and all following …
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Figure 4—source data 1
5 week old Atxn1154Q/2Q KI Inferior Olive enrichment chart for EnrichmentMap (FDR p < 0.05).
- https://doi.org/10.7554/eLife.39981.027
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Figure 4—source data 2
5 week old Atxn1154Q/2Q KI cerebellum enrichment chart edited for EnrichmentMap (nominal p-value < 0.01).
- https://doi.org/10.7554/eLife.39981.028
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Figure 4—source data 3
12 week old Atxn1154Q/2Q KI inferior olive enrichment chart for EnrichmentMap.
- https://doi.org/10.7554/eLife.39981.029
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Figure 4—source data 4
12 week old Atxn1154Q/2Q KI cerebellum enrichment chart for EnrichmentMap.
- https://doi.org/10.7554/eLife.39981.030
Figure 4—figure supplement 1
Minimal gene overlap in the 5 week old Atxn1154Q/2Q KI cerebellum and inferior olive in the cross-tissue comparison.
(A) Schematic of the cross-tissue comparison in the 5 week old Atxn1154Q/2Q KI inferior olive and cerebellum. (B) Total number of differentially regulated genes in the Atxn1154Q/2Q KI inferior olive …
Figure 5 with 3 supplements
The Defense Response is enriched in both the 12 week old Atxn1154Q/2Q KI inferior olive and ATXN1-82Q Tg inferior olive.
(A) ATXN1 mRNA expression in the 12 week old ATXN1-82Q Tg cerebellum and inferior olive (***p < 0.001, t-test; n = 3 males per genotype). (B) PolyQ-expanded ATXN1 is present in the cerebellum, but …
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Figure 5—source data 1
5 week old ATXN1-82Q Tg inferior olive functional annotation clustering analysis.
- https://doi.org/10.7554/eLife.39981.035
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Figure 5—source data 2
12 week old ATXN1-82Q Tg inferior olive functional annotation clustering.
- https://doi.org/10.7554/eLife.39981.036
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Figure 5—source data 3
5 week old ATXN1-82Q Tg inferior olive enrichment chart for EnrichmentMap.
- https://doi.org/10.7554/eLife.39981.037
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Figure 5—source data 4
12 week old ATXN1-82Q Tg inferior olive enrichment chart for EnrichmentMap.
- https://doi.org/10.7554/eLife.39981.038
Figure 5—figure supplement 1
The majority of genes significantly altered in the 5 week ATXN1-82Q Tg inferior olive are up-regulated.
(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in ATXN1-82Q Tg inferior olive at 5 weeks of age (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). …
Figure 5—figure supplement 2
Differentially regulated genes identified in the 12 week ATXN1-82Q Tg inferior olive are associated with Blood Vessel Morphogenesis, Response to Organic Substance, and Defense Response-related pathways.
(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in the 12 week old ATXN1-82Q Tg inferior olive (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). (C…
Figure 5—figure supplement 3
Divergent gene expression changes and minimal biological pathway overlap in the temporal comparison of the ATXN1-82Q Tg inferior olive.
(A) Schematic of the time-point comparison in ATXN1-82Q Tg inferior olive. (B) Total number of differentially regulated genes in the ATXN1-82Q Tg inferior olive relative to appropriate WT controls …
Figure 6 with 4 supplements
Divergent gene expression changes and low biological pathway overlap between 12 week old ATXN1-82Q Tg inferior olive and cerebellum.
(A) Schematic of the cross-tissue comparison in ATXN1-82Q Tg mice. (B) Total number of differentially regulated genes that are common, and uniquely altered, in the inferior olive and cerebellum of …
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Figure 6—source data 1
5 week old ATXN1-82Q Tg cerebellum functional annotation enrichment analysis.
- https://doi.org/10.7554/eLife.39981.044
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Figure 6—source data 2
12 week old ATXN1-82Q Tg functional annotation clustering enrichment analysis.
- https://doi.org/10.7554/eLife.39981.045
Figure 6—figure supplement 1
Chemical Synaptic Transmission and Regulation of Cell Communication are the most enriched pathways in the 5 week ATXN1-82Q Tg cerebellum.
(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in ATXN1-82Q Tg cerebellum at 5 weeks of age (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). (C) …
Figure 6—figure supplement 2
Pathways related to Synaptic Signaling are enriched in the 12 week ATXN1-82Q Tg cerebellum.
(A) Illustration of the tissue examined. (B) Total number of up- and down-regulated genes in ATXN1-82Q Tg cerebellum at 12 weeks of age (FDR p-value < 0.05; n = 3 males per genotype for RNA-seq). (C)…
Figure 6—figure supplement 3
Common gene expression changes and pathway enrichment in the 5 and 12 week ATXN1-82Q Tg cerebellum.
(A) Schematic of the time-point comparison in ATXN1-82Q Tg cerebellum. (B) Total number of differentially regulated genes in the ATXN1-82Q Tg cerebellum relative to appropriate WT controls that are …
Figure 6—figure supplement 4
Divergent gene expression changes and pathway enrichment in the 5 week ATXN1-82Q Tg inferior olive and cerebellum.
(A) Schematic of the cross-tissue comparison in ATXN1-82Q Tg inferior olive and cerebellum at 5 weeks of age. (B) Total number of differentially regulated genes in the ATXN1-82Q Tg cerebellum and …
Figure 7
Genes commonly regulated in the cerebellum across 12 week old SCA1 mouse models are altered in opposing directions.
(A) Schematic of the cross-model comparison in 12 week old Atxn1154Q/2Q KI and ATXN1-82Q Tg cerebellum. (B) Number and directionality of shared differentially regulated genes in the Atxn1154Q/2Q KI …
Figure 8
Summary of biological and molecular pathway enrichment across SCA1 mouse models in each brain region over time.
Differentially regulated genes in the 5 week old inferior olive and cerebellum from both SCA1 mouse models are associated with specific biological and molecular pathways, a subset of which are …
Tables
Table 1
Enrichment for Irf7 binding site DNA motifs in up-regulated 12 week old Atxn1154Q/2Q inferior olive genes.
https://doi.org/10.7554/eLife.39981.013| Brain region | Motif occurrence | Down-Regulated | Shuffled | p-value | q-value |
|---|---|---|---|---|---|
| Inferior Olive | Present | 32 | 26 | 0.468 | 0.123 |
| Absent | 128 | 134 | |||
| Brain Region | Motif Occurrence | Up-Regulated | Shuffled | p-value | q-value |
| Inferior Olive | Present | 27 | 2 | 8.8e-9 | 9.2e-9 |
| Absent | 17 | 42 |
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FDR p-value calculated using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q 5%).
Table 2
Enrichment for Cic binding site DNA motifs in down- and up-regulated 12 week old Atxn1154Q/2Q inferior olive and cerebellum.
https://doi.org/10.7554/eLife.39981.024| Cic DNA motif | Brain region | Motif occurrence | Down-Regulated | Shuffled | p-value | q-value |
|---|---|---|---|---|---|---|
| TGAATGAA | Inferior Olive | Present | 31 | 19 | 0.090 | 0.095 |
| Absent | 129 | 141 | ||||
| Cerebellum | Present | 126 | 57 | 5.6e-8 | 2.9e-7 | |
| Absent | 604 | 673 | ||||
| TGAATGGA | Inferior Olive | Present | 35 | 25 | 0.197 | 0.148 |
| Absent | 125 | 135 | ||||
| Cerebellum | Present | 157 | 104 | 3.7e-4 | 9.7e-4 | |
| Absent | 573 | 626 | ||||
| Cic DNA Motif | Brain Region | Motif Occurrence | Up-Regulated | Shuffled | p-value | q-value |
| TGAATGAA | Inferior Olive | Present | 10 | 4 | 0.143 | 0.125 |
| Absent | 34 | 40 | ||||
| Cerebellum | Present | 32 | 19 | 0.073 | 0.095 | |
| Absent | 181 | 194 | ||||
| TGAATGGA | Inferior Olive | Present | 13 | 2 | 0.003 | 0.005 |
| Absent | 31 | 42 | ||||
| Cerebellum | Present | 38 | 29 | 0.287 | 0.188 | |
| Absent | 175 | 184 |
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FDR p-value calculated using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q 5%).
Table 3
Enrichment for Cic binding site DNA motifs in down- and up-regulated genes from 12 week old ATXN1-82Q Tg inferior olive and cerebellum.
https://doi.org/10.7554/eLife.39981.046| Cic DNA motif | Brain region | Motif occurrence | Down-Regulated | Shuffled | p-value | q-value |
|---|---|---|---|---|---|---|
| TGAATGAA | Inferior Olive | Present | 12 | 1 | 0.002 | 0.002 |
| Absent | 56 | 67 | ||||
| Cerebellum | Present | 90 | 48 | 1.9e-4 | 4.0e-4 | |
| Absent | 510 | 552 | ||||
| TGAATGGA | Inferior Olive | Present | 19 | 13 | 0.312 | 0.187 |
| Absent | 49 | 55 | ||||
| Cerebellum | Present | 110 | 86 | 0.073 | 0.061 | |
| Absent | 490 | 514 | ||||
| Cic DNA Motif | Brain Region | Motif Occurrence | Up-Regulated | Shuffled | p-value | q-value |
| TGAATGAA | Inferior Olive | Present | 10 | 7 | 0.606 | 0.318 |
| Absent | 60 | 63 | ||||
| Cerebellum | Present | 97 | 51 | 4.9e-5 | 2.1e-4 | |
| Absent | 366 | 412 | ||||
| TGAATGGA | Inferior Olive | Present | 15 | 7 | 0.103 | 0.072 |
| Absent | 55 | 63 | ||||
| Cerebellum | Present | 96 | 59 | 0.002 | 0.002 | |
| Absent | 367 | 404 |
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FDR p-value calculated using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli (Q 5%).
Key resources table
| Reagent type | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, or strain background (Mus musculus, C57BL/6J) | Atxn1154Q/2Q KI | PMID:12086639 | RRID:MGI:2429435, backcrossed in house on C57BL/6J background | |
| Strain, or strain background (Mus musculus, FVB/NJ) | ATXN1-82Q Tg | PMID:7553854 | RRID:MGI:2447854 backcrossed in house on FVB/NJ background | |
| Strain, or strain background (Mus musculus, C57BL/6J) | C57BL/6J | The Jackson Lab | Stock: 000664 | |
| Strain, or strain background (Mus musculus, FVB/NJ) | FVB/NJ | The Jackson Lab | Stock: 001800 | |
| Cell line (Mus musculus) | BV2 | A kind gift from Dr. Katerina Akassoglou (Adams et al., 2007) | RRID: CVCL_0182 | |
| Transfected construct | human Flag-ATXN1 82Q | A kind gift from Dr. Huda Zoghbi | Addgene Plasmid #33237 | |
| Transfected construct | mammalian expression vector pcDNA3.1+ | Invitrogen (ThermoFisher Scientific) | Cat. # V790-20 | |
| antibody | rabbit polyclonal anti-Atxn111750 | a kind gift from Huda Zoghbi (Servadio et al., 1995) | RRID:AB_2721278 | (1:1,000) WB |
| Antibody | mouse monoclonal anti-Calbindin | Sigma | Cat. #: C9848 RRID:AB_476894 | (1:2,000) IHC |
| Antibody | mouse monoclonal anti-Gapdh | Sigma | Cat. #G8795 RRID:AB_1078991 | (1:10,000) WB |
| Antibody | chicken polyclonal anti-Gfap | Abcam | Cat. # ab4674 RRID:AB_304558 | (1:1,000) IHC |
| Antibody | rabbit polyclonal anti-Iba1 | Wako | Cat. # 019–19741 RRID:AB_839504 | (1:2,000) IHC |
| Antibody | goat polyclonal anti-chicken Alexa555 | ThermoFisher | Cat. #A21437 RRID:AB_2535858 | (1:500) IHC |
| Antibody | goat polyclonal anti-mouse Alexa488 | ThermoFisher | Cat. # A11001 RRID:AB_2534069 | (1:500) IHC |
| Antibody | goat polyclonal anti-rabbit Alexa568 | ThermoFisher | Cat. #A11011 RRID:AB_143157 | (1:500) IHC |
| Antibody | donkey anti-rabbit | GE Healthcare | Cat. #NA934 RRID:AB_772206 | (1:4,000) WB |
| Antibody | anti-mouse | GE Healthcare | Cat. #NXA931 RRID:AB_772209 | (1:4,000) WB |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | Cat #74106 | |
| Commercial assay or kit | iScript cDNA Synthesis Kit | Bio-Rad | Cat. #1708891 | |
| Commercial assay or kit | iTaq Universal Probes Supermix | Bio-Rad | Cat. #1725131 | |
| Commercial assay or kit | Pierce BCA Protein Assay | ThermoFisher | Cat. #23225 | |
| Commercial assay or kit | Amaxa Cell Line Nucleofector Kit T | Lonza | Cat. #VCA-1002 | |
| Other | DAPI | Vector Laboratories | Cat. #H-1500 RRID:AB_2336788 | |
| Other | Actb probe | Applied Biosystems | Cat. #4352932E | |
| Other | ATXN1 probe | Applied Biosystems | Cat. # 4331182 Hs00165656_m1 | |
| Other | Calb1 probe | Applied Biosystems | Cat. #4331182 Mm00486647_m1 | |
| Other | Gapdh probe | Applied Biosystems | Cat. #4352933E | |
| Other | Ifitm3 probe | Applied Biosystems | Cat. # 4331182 Mm00847057_s1 | |
| Other | Ifr7 probe | Applied Biosystems | Cat. #4331182 Mm00516793_g1 | |
| Other | Oasl2 probe | Applied Biosystems | Cat. # 4331182 Mm01201449_m1 | |
| Other | Stat1 probe | Applied Biosystems | Cat. # 4331182 Mm01257286_m1 | |
| Software, algorithm | Ingenuity Pathway Analysis (IPA) (Spring Release 2018) | http://www.ingenuity.com/products/pathways_analysis.html | RRID:SCR_008653 | |
| Software, algorithm | NIH DAVID v6.8 | http://david.abcc.ncifcrf.gov/ | RRID:SCR_001881 | |
| Software, algorithm | TopHat v2.1.0 | http://tophat.cbcb.umd.edu/ | RRID:SCR_013035 | |
| Software, algorithm | Cufflinks v2.2.1 | http://cole-trapnell-lab.github.io/cufflinks/ | RRID:SCR_014597 | |
| Software, algorithm | Bowtie2 v2.1.0 | http://bowtie-bio.sourceforge.net/bowtie2/index.shtml | RRID:SCR_016368 | |
| Software, algorithm | FastQC v0.11.3 | http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ | RRID:SCR_014583 | |
| Software, algorithm | Cummerbund | http://compbio.mit.edu/cummeRbund/index.html | RRID:SCR_014568 | |
| Software, algorithm | Bioconductor R with heatmap.2 gplots package | https://www.rdocumentation.org/packages/gplots/versions/3.0.1/topics/heatmap.2 | ||
| Software, algorithm | R project for statistical computing v3.3.3 | http://www.r-project.org/ | RRID:SCR_001905 | |
| Software, algorithm | MEME Suite - Motif-based sequence analysis tools | http://meme-suite.org/ | RRID:SCR_001783 | |
| Software, algorithm | EnrichmentMap | http://baderlab.org/Software/EnrichmentMap | RRID:SCR_016052 | |
| Software, algorithm | Cytoscape v3.5.1 | http://cytoscape.org | RRID:SCR_003032 | |
| Software, algorithm | GraphPad Prism | http://www.graphpad.com/ | RRID:SCR_002798 | |
| Software, algorithm | ImageJ | https://imagej.net/ | RRID:SCR_003070 |
Additional files
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Supplementary file 1
5 week old Atxn1154Q/2Q KI inferior olive IPA canonical pathway enrichment.
- https://doi.org/10.7554/eLife.39981.049
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Supplementary file 2
5 week old Atxn1154Q/2Q KI inferior olive IPA predicted upstream regulators.
- https://doi.org/10.7554/eLife.39981.050
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Supplementary file 3
Upstream regulators for differentially regulated 12 week old Atxn1154Q/2Q KI cerebellum genes.
- https://doi.org/10.7554/eLife.39981.051
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Supplementary file 4
5 week old ATXN1-82Q Tg inferior olive IPA canonical pathways.
- https://doi.org/10.7554/eLife.39981.052
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Supplementary file 5
12 week old ATXN1-82Q Tg inferior olive upstream regulators predicted in IPA.
- https://doi.org/10.7554/eLife.39981.053
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Supplementary file 6
RNA integrity numbers (RIN) for samples used in RNA-seq analysis.
- https://doi.org/10.7554/eLife.39981.054
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Transparent reporting form
- https://doi.org/10.7554/eLife.39981.055
