Gene Expression
Nebulas
Gene Expression NebulasSummary: There is currently little information about how individual cell types contribute to Alzheimer’s disease (AD). Here, we applied single-nucleus RNA-seq (snRNA-seq) on the entorhinal cortex from control and AD brains of twelve individuals, yielding a total of 13,214 high quality nuclei. We detail cell-type-specific gene expression patterns, unveiling how transcriptional changes in specific cell subpopulations contribute to AD. We report that the AD risk gene, APOE, is specifically repressed in AD oligodendrocyte progenitor cells and astrocyte subpopulations, and upregulated in an AD-specific microglial subopulation. Integrating transcription factor regulatory modules with AD risk loci revealed drivers of cell-type-specific fate transitions towards AD. For example, transcription factor EB, a master regulator of lysosomal function, regulates multiple disease genes in a specific AD astrocyte sub-population. These results provide insights into the coordinated control of AD risk genes and their cell-type specific contribution to disease susceptibility and are available at http://adsn.ddnetbio.com.
Overall Design: Single Nuclei RNA sequencing of 8 10x libraries with each library containing 2 individuals (n =16)
| Strategy: |
|
| Species: |
|
| Tissue: |
|
| Healthy Condition: |
|
| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | Entorhinal cortex tissue from post-mortem Alzheimer's disease and non-disease aged matched individuals was obtained from the Victorian Brain Bank (Ethics Approval for patient tissue banking and consent: University of Melbourne HREC Approval No.: 1545740; approval for transcriptomic analysis using banked tissue Monash University MUHREC 2016-0554; patient demographics in Supplementary Table 1). Nuclei isolation was carried out using the Nuclei Isolation Kit: Nuclei EZ Prep (Sigma, #NUC101) as described in 9. Briefly, tissue samples were homogenized using a glass dounce grinder in 2 ml of ice-cold EZ PREP and incubated on ice for 5 min. Centrifuged nuclei (500 xg, 5 min and 4℃) were washed in ice-cold EZ PREP buffer, and Nuclei Suspension Buffer (NSB; consisting of 1xPBS, 1% (w/v) BSA and 0.2 U/μl RNase inhibitor (Clontech, #2313A). Isolated nuclei were resuspended in NSB to 106 nuclei per 400 μl), filtered through a 40 μm cell strainer and counted with Trypan blue. Nuclei enriched in Nuclei Suspension Buffer were stained with DAPI (1:1000) for nuclei isolation using the Influx cell sorter (BD Biosciences, Franklin Lakes, NJ; 70 μm nozzle, 21-22 psi). Nuclei were defined as DAPI+ singlets. Sorted nuclei were counted twice prior to loading onto the 10X Chromium (10X Genomics). |
| Library Construction Protocol: | Library construction was performed using the Chromium Single Cell 3’ Library & Gel Bead Kit v2 (10X Genomics, #PN-120237) with 18 cDNA pre-amplification cycles and sequencing on one high-output lane of the NextSeq 500 (Illumina). Our sequencing saturation was high, ranging from 83.2% to 97.4%. |
| Molecule Type: | poly(A)+ RNA |
| Library Source: | |
| Library Layout: | PAIRED |
| Library Strand: | Forward |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Specific |
| Data Resource | GEN Sample ID | GEN Dataset ID | Project ID | BioProject ID | Sample ID | Sample Name | BioSample ID | Sample Accession | Experiment Accession | Release Date | Submission Date | Update Date | Species | Race | Ethnicity | Age | Age Unit | Gender | Source Name | Tissue | Cell Type | Cell Subtype | Cell Line | Disease | Disease State | Development Stage | Mutation | Phenotype | Case Detail | Control Detail | Growth Protocol | Treatment Protocol | Extract Protocol | Library Construction Protocol | Molecule Type | Library Layout | Strand-Specific | Library Strand | Spike-In | Strategy | Platform | Instrument Model | Cell Number | Reads Number | Gbases | AvgSpotLen1 | AvgSpotLen2 | Uniq Mapping Rate | Multiple Mapping Rate | Coverage Rate |
|---|