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Status |
Public on Jan 14, 2023 |
Title |
E16 DZ, scRNAseq |
Sample type |
SRA |
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Source name |
Somite
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Organism |
Sus scrofa |
Characteristics |
tissue: Somite developmental stage: embryonic day (E) 16 breed: DurocxTibetan pigs (DZ)
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Extracted molecule |
total RNA |
Extraction protocol |
Somite tissues of ZZ and DZ at E18, E21, and myotome tissues at E28 were isolated and transferred to RPMI1640 medium (GIBCO, 11875093) containing 10% Fetal Bovine Serum (HyClone, SH30070.03) on ice. Then, the tissues were washed with PBS three times and transferred to pre-warmed digestion medium containing 100 μg/mL DNase Ⅰ (Sigma-Aldrich, DN25) and 0.1 g/mL Collagenase I (Sigma, C2674) in RPMI1640. Tissues were shaken vigorously for 30 s and further incubated at 37°C for 30 to 40 minutes in an incubator with general shaking every 6 minutes to release cells. Released cells passed through a 70 μm cell strainer (BD, 352350) and were collected in 15 mL tubes. Sample viability was assessed via Trypan Blue (ThermoFisher) and an automatic cell counter (Countstar). Cells derived from different embryo ages and pigs were sorted and processed independently in all experiments. Thus, each cell can be traced back to the embryos and tissues it came from. Droplet-based scRNA-seq datasets were produced using a Chromium system (10x Genomics, PN120263) following the manufacturer’s instruction. After droplet generation, samples were transferred onto a pre-chilled 8-well tube (Eppendorf), and reverse transcription was performed using a Veriti 96-well thermal cycler (Thermo Fisher). After the reverse transcription, cDNA was recovered using Recovery Agent provided by 10x Genomics, followed by a Silane DynaBead clean-up (Thermo Fisher) as outlined in the user guide. Purified cDNA was amplified for 12 cycles before being cleaned up using SPRIselect beads (Beckman). Samples were diluted at a ratio of 1:4 and run on a Bioanalyzer (Agilent Technologies) to determine cDNA concentration. cDNA libraries were prepared as outlined by the Single Cell 3′Reagent Kits v3 user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration (as recommended by 10X Genomics).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
10x Genomics
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Data processing |
Raw sequencing data were aligned to the pig gene expression reference Sscrofa11 (the 10× reference database was prepared with the gene annotation gff3 dataset v11.1.98 and genomic fasta dataset v11.1.98, which were downloaded from Ensemble). The CellRanger (10× Genomics) analysis pipeline was used to generate a digital gene expression matrix from these data. The raw digital gene expression matrix (UMI counts per gene per cell) was filtered, normalized, and clustered using R [v3.5.2, https://www.R-project.org/]. Cell and gene filtering was performed as follows: cells with fewer than 200 genes detected, or more than 30,000 UMIs and cells containing greater than 10% of reads from mitochondrial genes were removed. Assembly: Sscrofa11 Supplementary files format and content: Cell Ranger output files (barcodes.tsv, features.tsv, matrix.mtx)
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Submission date |
Jun 24, 2022 |
Last update date |
Jan 14, 2023 |
Contact name |
Delin Mo |
E-mail(s) |
wangxy635@mail2.sysu.edu.cn
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Organization name |
Sun Yat-sen University
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Street address |
Xingang Road
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City |
Guangzhou |
ZIP/Postal code |
510006 |
Country |
China |
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Platform ID |
GPL26351 |
Series (2) |
GSE206911 |
Single-cell RNA gene expression profiles of the somites and myotomes at embryonic stages E18, E21, and E28 from Tibetan pigs (ZZ) and Duroc x Tibetan pigs (DZ) |
GSE206914 |
Somites and myotomes at embryonic stages E18, E21, and E28 from Tibetan pigs (ZZ) and Duroc x Tibetan pigs (DZ) |
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Relations |
BioSample |
SAMN29406401 |
SRA |
SRX15890961 |
Supplementary file |
Size |
Download |
File type/resource |
GSM6266742_Y20SS00149-barcodes.tsv.gz |
57.5 Kb |
(ftp)(http) |
TSV |
GSM6266742_Y20SS00149-features.tsv.gz |
173.9 Kb |
(ftp)(http) |
TSV |
GSM6266742_Y20SS00149-matrix.mtx.gz |
75.6 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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