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| Status |
Public on Aug 25, 2021 |
| Title |
The skin tissue of cashmere is taken from scapularis of healthy Liaoning cashmere goats 2 |
| Sample type |
SRA |
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| Source name |
skin tissue
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| Organism |
Capra hircus |
| Characteristics |
growth cycle: anagen
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| Growth protocol |
Skin tissue was obtained from healthy Liaoning Cashmere Goats.Place a sterile RNase-free culture dish containing an appropriate amount of calcium-free and magnesium-free 1 × PBS on ice, the tissue was transferred into the culture dish and cut it into 0.5 mm2 pieces, the tissues were washed with 1 × PBS, and remove as many non-purpose tissues as possible such as blood stains and fatty layers.Tissue s were dissociated into single cells in dissociation solution in 37 ℃ water bath with shaking for 20 min at 100 rpm. Digestion was terminated with 1× PBS containing 10% fetal bovine serum（FBS，V/V）, then pipetting 5-10 times with a Pasteur pipette。The resulting cell suspension was filtered by passing through 70-30um stacked cell strainer and centrifuged at 300g for 5 min at 4 °C. The cell pellet was resuspended in 100ul 1× PBS (0.04% BSA) and added with 1 ml 1× red blood cell lysis buffer（MACS 130-094-183, 10×）and incubated at room temperature or on ice for 2-10 min to lyse remaining red blood cells. After incubation, the suspension was centrifuged at 300g for 5 min at room temperature. The suspension was resuspended in 100 μl Dead Cell Removal MicroBeads(MACS 130-090-101) and remove dead cells using Miltenyi ® Dead Cell Removal Kit (MACS 130-090-101). Then the suspension was resuspended in 1× PBS(0.04% BSA) and centrifuged at 300 g for 3 min at 4 °C（repeat twice）. The cell pellet was resuspended in 50 μl of 1× PBS (0.04% BSA). The overall cell viability was confirmed by trypan blue exclusion , which needed to be above 85%, single cell suspensions were counted using a haemocytometer/ Countess II Automated Cell Counter and concentration adjusted to 700-1200 cells/μl.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single-cell suspensions were loaded to 10x Chromium to capture 5000 single cell according to the manufacturer’s instructions of 10X Genomics Chromium Single-Cell 3’kit (V3) .The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run,150bp) by LC-Bio Technology co.ltd., (HangZhou,China) at a minimum depth of 20,000 reads per cell.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software（version2.20）. Sample demultiplexing, barcode processing and single-cell 3’gene counting by using the Cell Ranger pipeline(https://support.10xgenomics.com/single-cell-geneexpression/ software/pipelines/latest/what-is-cell-ranger，version 5.0.1) and scRNA-seq data were aligned to Ensembl genome GRCh38/GRCm38 reference genome, a total of 30,000 single cell captured from 5 healthly donors and 5 patients were processed using 10X Genomics Chromium Single Cell 3’Solution. The Cell Ranger output was loaded into Seurat(version 3.1.1) be used to Dimensional reduction, clustering, and analysis of scRNA-seq data. Overall, 25,000 cells passed the quality control threshold : all genes expressed in less than three cells （default parameters：1 cell）were removed、number of genes expressed per cell > 500 as low and <5000 as high cut-off、UMI counts less than 500、the percent of mitochondrial-DNA derived gene-expression <25%.
To visualize the data, we further reduced the dimensionality of all 18,339 cells using Seurat and used t-SNE to project the cells into 2D space. The steps includes:1、Using the LogNormalize method of the "Normalization" function of the Seurat software to calculated the expression value of genes; 2、PCA (Principal component analysis) analysis was performed using the normalized expression value, Within all the PCs, the top 10 PCs were used to do clustering and t-SNE analysis；3、To find clusters, selecting weighted Shared Nearest Neighbor (SNN) graph-based clustering method. Marker genes for each cluster were identified with the Wilcoxon rank-sum test （default parameters is"bimod" : Likelihood-ratio test）with default parameters via the FindAllMarkers function in Seurat. This selects markers genes which are expressed in more than 10% of the cells in a cluster and average log(Fold Change) of greater than 0.25（default parameters：0.26）.
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| Submission date |
Aug 19, 2021 |
| Last update date |
Aug 25, 2021 |
| Contact name |
Ying Ze Wang |
| E-mail(s) |
wangzeying2012@syau.edu.cn
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| Phone |
15998362195
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| Organization name |
沈阳农业大学
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| Street address |
120 Dongling Road, Shenhe District, Shenyang
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| City |
Shenyang |
| State/province |
Liaoning |
| ZIP/Postal code |
110866 |
| Country |
China |
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| Platform ID |
GPL27106 |
| Series (1) |
| GSE182474 |
Single-cell sequencing reveals differential cell types in skin tissues of Liaoning Cashmere Goats and key genes related potentially to the fineness of cashmere fiber |
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| Relations |
| BioSample |
SAMN20863834 |
| SRA |
SRX11828768 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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