Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe + microglia/macrophage after deeper cerebral hypoperfusion in mice
Abstract Background Ambient RNAs contamination in single-nuclei RNA sequencing (snRNA-seq) is a challenging problem, but the consequences of ambient RNAs contamination of damaged and/or diseased tissues are poorly understood. Cognitive impairments and white/gray matter injuries are characteristic of...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2023-06-01
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| Series: | Journal of Neuroinflammation |
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| Online Access: | https://doi.org/10.1186/s12974-023-02831-9 |
| _version_ | 1797789653622849536 |
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| author | Yuan Zhang Jinyun Tan Kai Yang Weijian Fan Bo Yu Weihao Shi |
| author_facet | Yuan Zhang Jinyun Tan Kai Yang Weijian Fan Bo Yu Weihao Shi |
| author_sort | Yuan Zhang |
| collection | DOAJ |
| description | Abstract Background Ambient RNAs contamination in single-nuclei RNA sequencing (snRNA-seq) is a challenging problem, but the consequences of ambient RNAs contamination of damaged and/or diseased tissues are poorly understood. Cognitive impairments and white/gray matter injuries are characteristic of deeper cerebral hypoperfusion mouse models induced by bilateral carotid artery stenosis (BCAS), but the molecular mechanisms still need to be further explored. More importantly, the BCAS mice can also offer an excellent model to examine the signatures of ambient RNAs contamination in damaged tissues when performing snRNA-seq. Methods After the sham and BCAS mice were established, cortex-specific single-nuclei libraries were constructed. Single-nuclei transcriptomes were described informatically by the R package Seurat, and ambient RNA markers of were identified in each library. Then, after removing ambient RNAs in each sample using the in silico approaches, the combination of CellBender and subcluster cleaning, single-nuclei transcriptomes were reconstructed. Next, the comparison of ambient RNA contamination was performed using irGSEA analysis before and after the in silico approaches. Finally, further bioinformatic analyses were performed. Results The ambient RNAs are more predominant in the BCAS group than the sham group. The contamination mainly originated from damaged neuronal nuclei, but could be reduced largely using the in silico approaches. The integrative analysis of cortex-specific snRNA-seq data and the published bulk transcriptome revealed that microglia and other immune cells were the primary effectors. In the sequential microglia/immune subgroups analysis, the subgroup of Apoe + MG/Mac (microglia/macrophages) was identified. Interestingly, this subgroup mainly participated in the pathways of lipid metabolism, associated with the phagocytosis of cell debris. Conclusions Taken together, our current study unravels the features of ambient RNAs in snRNA-seq datasets under diseased conditions, and the in silico approaches can effectively eliminate the incorrected cell annotation and following misleading analysis. In the future, snRNA-seq data analysis should be carefully revisited, and ambient RNAs removal needs to be taken into consideration, especially for those diseased tissues. To our best knowledge, our study also offers the first cortex-specific snRNA-seq data of deeper cerebral hypoperfusion, which provides with novel therapeutic targets. |
| first_indexed | 2024-03-13T01:53:44Z |
| format | Article |
| id | doaj.art-b115d0e596d54ae3a22f42a6602c2592 |
| institution | Directory Open Access Journal |
| issn | 1742-2094 |
| language | English |
| last_indexed | 2024-03-13T01:53:44Z |
| publishDate | 2023-06-01 |
| publisher | BMC |
| record_format | Article |
| series | Journal of Neuroinflammation |
| spelling | doaj.art-b115d0e596d54ae3a22f42a6602c25922023-07-02T11:21:41ZengBMCJournal of Neuroinflammation1742-20942023-06-0120112310.1186/s12974-023-02831-9Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe + microglia/macrophage after deeper cerebral hypoperfusion in miceYuan Zhang0Jinyun Tan1Kai Yang2Weijian Fan3Bo Yu4Weihao Shi5Department of Vascular Surgery, Shanghai Pudong Hospital, Fudan University Pudong Medical CenterDepartment of Vascular Surgery, Huashan Hospital of Fudan UniversityDepartment of Vascular Surgery, Shanghai Pudong Hospital, Fudan University Pudong Medical CenterDepartment of Vascular Surgery, Shanghai Pudong Hospital, Fudan University Pudong Medical CenterDepartment of Vascular Surgery, Shanghai Pudong Hospital, Fudan University Pudong Medical CenterDepartment of Vascular Surgery, Huashan Hospital of Fudan UniversityAbstract Background Ambient RNAs contamination in single-nuclei RNA sequencing (snRNA-seq) is a challenging problem, but the consequences of ambient RNAs contamination of damaged and/or diseased tissues are poorly understood. Cognitive impairments and white/gray matter injuries are characteristic of deeper cerebral hypoperfusion mouse models induced by bilateral carotid artery stenosis (BCAS), but the molecular mechanisms still need to be further explored. More importantly, the BCAS mice can also offer an excellent model to examine the signatures of ambient RNAs contamination in damaged tissues when performing snRNA-seq. Methods After the sham and BCAS mice were established, cortex-specific single-nuclei libraries were constructed. Single-nuclei transcriptomes were described informatically by the R package Seurat, and ambient RNA markers of were identified in each library. Then, after removing ambient RNAs in each sample using the in silico approaches, the combination of CellBender and subcluster cleaning, single-nuclei transcriptomes were reconstructed. Next, the comparison of ambient RNA contamination was performed using irGSEA analysis before and after the in silico approaches. Finally, further bioinformatic analyses were performed. Results The ambient RNAs are more predominant in the BCAS group than the sham group. The contamination mainly originated from damaged neuronal nuclei, but could be reduced largely using the in silico approaches. The integrative analysis of cortex-specific snRNA-seq data and the published bulk transcriptome revealed that microglia and other immune cells were the primary effectors. In the sequential microglia/immune subgroups analysis, the subgroup of Apoe + MG/Mac (microglia/macrophages) was identified. Interestingly, this subgroup mainly participated in the pathways of lipid metabolism, associated with the phagocytosis of cell debris. Conclusions Taken together, our current study unravels the features of ambient RNAs in snRNA-seq datasets under diseased conditions, and the in silico approaches can effectively eliminate the incorrected cell annotation and following misleading analysis. In the future, snRNA-seq data analysis should be carefully revisited, and ambient RNAs removal needs to be taken into consideration, especially for those diseased tissues. To our best knowledge, our study also offers the first cortex-specific snRNA-seq data of deeper cerebral hypoperfusion, which provides with novel therapeutic targets.https://doi.org/10.1186/s12974-023-02831-9Ambient RNAs contaminationDeeper chronic cerebral hypoperfusionNeuroinflammationApoe + MG/Mac |
| spellingShingle | Yuan Zhang Jinyun Tan Kai Yang Weijian Fan Bo Yu Weihao Shi Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe + microglia/macrophage after deeper cerebral hypoperfusion in mice Journal of Neuroinflammation Ambient RNAs contamination Deeper chronic cerebral hypoperfusion Neuroinflammation Apoe + MG/Mac |
| title | Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe + microglia/macrophage after deeper cerebral hypoperfusion in mice |
| title_full | Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe + microglia/macrophage after deeper cerebral hypoperfusion in mice |
| title_fullStr | Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe + microglia/macrophage after deeper cerebral hypoperfusion in mice |
| title_full_unstemmed | Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe + microglia/macrophage after deeper cerebral hypoperfusion in mice |
| title_short | Ambient RNAs removal of cortex-specific snRNA-seq reveals Apoe + microglia/macrophage after deeper cerebral hypoperfusion in mice |
| title_sort | ambient rnas removal of cortex specific snrna seq reveals apoe microglia macrophage after deeper cerebral hypoperfusion in mice |
| topic | Ambient RNAs contamination Deeper chronic cerebral hypoperfusion Neuroinflammation Apoe + MG/Mac |
| url | https://doi.org/10.1186/s12974-023-02831-9 |
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