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| Status |
Public on Oct 06, 2021 |
| Title |
AR1_MI28_P56_7995_RZ |
| Sample type |
SRA |
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| Source name |
7995RZ
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| Organism |
Sus scrofa |
| Characteristics |
tissue: Heart
arp1 (yes/no): yes
mip28 (yes/no): yes
harvest postnatal day (p): P56
heart zone: Remote zone
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| Treatment protocol |
Briefly, pigs were anesthetized with isoflurane and placed in a dorsal recumbent position on a heating pad, and a median sternotomy was performed to expose the heart. Then, the sternum was re-approximated, the chest was closed in layers, and the air was evacuated from the mediastinum. After surgery, the animals were maintained in a temperature-controlled incubator until old enough to maintain their body temperature. On P28, animals came back to the operating room, and the left anterior descending coronary artery distal to the second diagonal was ligated by a ligature. The chest was closed in layers, and animals were allowed to recover.
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| Growth protocol |
All experimental protocols were approved by The Institutional Animal Care and Use Committee 5 (IACUC) at the University of Alabama at Birmingham and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No 85-23). Prestage farm pigs at various ages were purchase from Prestage Farm Inc. (West Point, MS). All pigs younger than P14 were housed in an incubator at a temperature of ~850F and room air. Piglets were fed with bovine colostrum every 4 hours for the first 2 days of life, 1:1 colostrum/sow’s milk at day 3 of life, and sow’s milk thereafter. Animals were given supplemental iron at day 7.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tissues were cut while submerged in cold phosphate-buffered saline (PBS) or UW solution and washed to remove blood; then, the solution was replaced with 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). Tissues were cut into even 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 s, and more lysis buffer was added. The mixture was placed on ice for ten minutes, filtered with 100-μm and 70-μm strainers, and centrifuged for 5 min at 700 rcf and 4°C; then, the supernatant was removed, and the pellet was resuspended in 10 mL nuclei wash and resuspension buffer (1X 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 min at 700 rcf and 4°C. The supernatant was removed and the pellet was resuspended in 1 mL nuclei wash and resuspension buffer; then, the nuclei were centrifuged again for 5 min at 700 rcf and 4 °C, the supernatant was removed, and 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 min at 13000 g and 4 °C. All but 100 μL of the supernatant was removed, and nuclei wash and resuspension buffer was added. The nuclei were resuspended, 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 min, and then immediately processed via the 10× Genomics® Single-Cell Protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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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)
Data filter. 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. Cells 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.
Genome_build: Sscrofa11.1 premRNA
Supplementary_files_format_and_content: The process data is in file Cardiomyocyte_Seurat.Robj. This file contains both the metadata and processed expression. The file can be directly opened by R, with Seurat package installed.
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| Submission date |
Oct 04, 2021 |
| Last update date |
Oct 06, 2021 |
| 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 |
GPL22475 |
| 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 |
SAMN22047213 |
| SRA |
SRX12485971 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5610184_7995RZ_matrix.zip |
52.4 Mb |
(ftp)(http) |
ZIP |
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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