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| Status |
Public on Dec 22, 2022 |
| Title |
Cochlea_wildtype_P1_multiome_ATAC |
| Sample type |
SRA |
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| Source name |
Inner ear cochlea
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| Organism |
Mus musculus |
| Characteristics |
tissue: Inner ear cochlea
developmental stage: P1
strain: mixed (CD1, FVB/NJ, C57BL/6)
genotype: wildtype
treatment: no treatment
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| Treatment protocol |
No treatment.
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| Growth protocol |
Wildtype mice in a mixed background of CD1 and FVB/NJ, and C57BL/6 were bred.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cochlear tissue was dissected from P1 and P8 mice and enzymatically dissociated with a cocktail of 400µl of 0.25% Trypsin, 50µl of 1 mg/ml Dispase, and 50µl of 1 mg/ml collagenase for 20 minutes at 37°C. After incubation, the digested tissue was titurated 100 times using a small-bore 200µl pipette and centrifuged at 500x g for 5 minutes at 4°C. After centrifugation, the sample was processed according to the 10x Genomics protocol “Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing” (CG000375, Rev B). Briefly, the supernatant was decanted, and the cell pellet was resuspended in 1ml of NP-40 Lysis Buffer and incubated for 5 min on ice.
Nuclei were filtered through a 40 micron cell strainer, and centrifuged at 500x g for 5 minutes at 4°C. The supernatant was decanted, and nuclei were resuspended in 1ml of PBS + 2% BSA and incubated for 5 min at 4C. Nuclei were centrifuged at 500x g for 5 minutes at 4°C, resuspended in 100ul of 0.1x Lysis Buffer, and mixed with a pipet. Nuclei were incubated for 2 minutes on ice, followed by the addition of 1ml Wash Buffer, mixed and centrifuged at 500x g for 5 minutes at 4°C, and resuspended in the appropriate volume of Diluted Nuclei Buffer for input into the Single Cell Multiome ATAC + Gene Expression protocol (10x Genomics). For the P1 cochlea, 6,065 nuclei were loaded. For the P8 cochlea, 9,645 nuclei were loaded.
scMultiome ATAC + Gene Expression protocol (10x Genomics).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
P1 wildtype cochlea 10x Genomics single-cell multiome ATAC
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| Data processing |
Raw sequencing data from both RNA and ATAC libraries in fastq format were used as input into cellranger-arc count (10x Genomics, v2.0.0) for simultaneous alignment against the mouse mm10 genome.
The cellranger- arc output files ‘filtered_feature_bc_matrix.h5’ and ‘atac_fragments.tsv.gz’ were used as input into Seurat v4.1.0 for standard quality control pre-processing, resulting in 4,882 nuclei post-filtering for the P1 dataset, and 7,049 nuclei post-filtering for the P8 dataset. ATAC peaks were called using macs2 v2.1.2. Multiome datasets were clustered based on RNA, ATAC, and weighted-nearest neighbor (Hao et al., 2021) to generate UMAPs. This resulted in 20 clusters for the P1 dataset, and 19 clusters for the P8 dataset. Clusters were assigned cell types based on known cell markers.
Representative signal tracks were visualized in IGV v2.4.14. ATAC peaks were filtered by hair cell enhancers previously identified (Tao et al., 2021), and intersected to find common, P1-specific, and P8-specific regions. Heatmaps were generated using deepTools v3.2.0 computeMatrix and plotHeatmap.
Cell barcodes from clusters of interest (P1 GER, P8 GER, P1 IPh/BC, P8 IPh, and P8 BC) were used to extract ATAC reads belonging to each respective cell type and generate a pseudobulk ATAC dataset.
Peaks were called on the pseudobulk ATAC datasets and common peaks were used as input into DESeq2 v1.34.0 to scale the signal between P1 and P8 datasets.
Assembly: mm10
Supplementary files format and content: cellranger output of aligned reads as bam files. Converted to feature-barcode matrices of gene expression from individual cells. Raw matrices contain every barcode sequence detected, including background, non-cellular barcodes, and low quality cells. filtered contains only detected cellular barcodes with quality control cutoff.
Supplementary files format and content: Count and barcode data for every ATAC fragment is stored in the atac fragments file.
Supplementary files format and content: Gene expression matrices and ATAC were loaded into Seurat to perform WNN UMAP clustering and cluster identification. The published cluster identities are contained in the cluster annotation file.
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| Submission date |
Dec 21, 2022 |
| Last update date |
Dec 22, 2022 |
| Contact name |
Andrew K Groves |
| Organization name |
Baylor College of Medicine
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| Street address |
One Baylor Plaza, Baylor College of Medicine
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE182202 |
Single cell transcriptomic analysis of P8 and P15 mouse cochlea (control and three overexpression conditions) and simultaneous single cell transcriptomic and accessible chromatin analysis of P1 and P8 mouse cochlea (wildtype) |
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| Relations |
| BioSample |
SAMN32357955 |
| SRA |
SRX18815993 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6883296_P1_cochlea_wildtype_10x_scRNA_scATAC_atac_fragments.tsv.gz |
3.5 Gb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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