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| Status |
Public on Jun 10, 2022 |
| Title |
8179_BZ_MIp28-P35 |
| Sample type |
SRA |
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| Source name |
Heart
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| Organism |
Sus scrofa |
| Characteristics |
cell type: Cardiomyocyte (main focus) and other cardiac cell types (not being analyzed)
tissue: Heart
age: 35 day
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were cut while submerged in cold phosphate-buffered saline (PBS) or UW solution, washed to remove blood, transferred into 1 mL lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.1% Nonidet™ P40 Substitute, and 50 U/mL RNase inhibitor in DEPC-treated water), cut into smaller pieces, and aspirated with the lysis buffer into a 50-mL tube; then, 10 mL of lysis buffer was added, the tissues were ground for 20-30 seconds, and more lysis buffer was added. The mixture was placed on ice for 10 minutes, filtered with 100-μm and 70-μm strainers, and centrifuged for 5 minutes at 700 rcf and 4 °C; then, the supernatant was removed, and the pellet was resuspended in 10 mL nuclei wash and resuspension buffer (1× PBS, 1.0% bovine serum albumin [BSA], and 50 U/mL RNase inhibitor). The suspension was passed through a 40-μm strainer, and the nuclei were centrifuged again for 5 minutes at 700 rcf and 4 °C; then, the supernatant was removed, the pellet was resuspended in 1 mL nuclei wash and resuspension buffer, and the nuclei were centrifuged a third time for 5 minutes at 700 rcf and 4 °C. The supernatant was removed; then, the pellet was resuspended in 5 mL sucrose cushion buffer I (2.7 mL Nuclei PURE 2M Sucrose Cushion Solution and 300 μL Nuclei PURE Sucrose Cushion Buffer) and mixed with 10 mL sucrose buffer. The mixture was layered over 5 mL of sucrose cushion buffer in a second Eppendorf tube and then centrifuged for 60 minutes at 13000 g and 4 °C. All but 100 μL of the supernatant was removed, the nuclei were resuspended in nuclei wash and resuspension buffer, and the solution was passed through a 40-μm strainer; then, the nuclei concentration was determined with a cell counter or hemocytometer and adjusted to 1000 nuclei/μL
The nuclei were placed on ice, stained with propidium iodide for 5 minutes, and then immediately processed via the 10× Genomics® Single-Cell Protocol
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10x Genomics
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| Data processing |
Sample demultiplexing, barcode processing, and gene counting were performed with Cell Ranger Single-Cell Software v.3.10 (https://support.10xgenomics.com/single-cell-gene expression/software). Reads were aligned to the Sscrofa11.1 pre-mRNA reference genome [78], and the pre-mRNA portion of the reference genome was extracted for single-nuclei UMI mapping with Cell Ranger mkref pre-mRNA
Only confidently mapped reads with valid barcodes and UMIs were used to generate the gene-barcode matrix. Cross-sample integration and quality-control were performed with the Seurat R package. Doublets were identified by using Seurat’s standard pipeline (https://satijalab.org/seurat/v3.2/pbmc3k_tutorial.html), and barcodes were removed if they had fewer than 500 UMIs, more than 30000 UMIs, or >5% mitochondrial UMIs
Nuclei were removed if they had <200 detected genes or if >25% of the transcripts were from mitochondrial genes. Mitochondrial genes and other transcript identifiers were removed without mapping to the official gene symbols from later analyses. Data were normalized as directed in the online Seurat tutorial (https://satijalab.org/seurat/v3.2/pbmc3k_tutorial.html); total expression was multiplied by a factor of 10,000 and then log-transformed
Assembly: Sscrofa11.1 pre-mRNA
Supplementary files format and content: Cell ranger output format (10X) for each sample
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| Submission date |
Jun 09, 2022 |
| Last update date |
Jun 11, 2022 |
| Contact name |
Thanh Minh Nguyen |
| E-mail(s) |
thamnguy@uab.edu
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| Phone |
3174108458
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| Organization name |
University of Alabama at Birmingham
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| Department |
Department of Biomedical Engineering
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| Street address |
1670 University Blvd, Volker Hall G094
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| City |
Birmingham |
| State/province |
AL |
| ZIP/Postal code |
35233 |
| Country |
USA |
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| Platform ID |
GPL20983 |
| Series (1) |
| GSE185289 |
Single nucleus transcriptomics: Apical resection in newborn pigs extends the time-window of cardiomyocyte proliferation and myocardial regeneration |
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| Relations |
| BioSample |
SAMN28940002 |
| SRA |
SRX15651104 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6224110_8179_BZ_barcodes.tsv.gz |
30.7 Kb |
(ftp)(http) |
TSV |
| GSM6224110_8179_BZ_features.tsv.gz |
184.5 Kb |
(ftp)(http) |
TSV |
| GSM6224110_8179_BZ_matrix.mtx.gz |
41.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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