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| Status |
Public on Nov 30, 2023 |
| Title |
old naked mole-rat anti-CD3 sorted splenocyte short-read 10x scRNA-seq, replicate 3 |
| Sample type |
SRA |
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| Source name |
spleen
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| Organism |
Heterocephalus glaber |
| Characteristics |
tissue: spleen
cell type: mostly spleen T cells
age: 24 years
Sex: male
colony/strain: 5000
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| Extracted molecule |
total RNA |
| Extraction protocol |
Spleens were removed by dissection, transferred to a sterile petri dish containing PBS with 5% FBS, and minced using surgical blades. Spleen fragments were then ground through 100 μm and 40 μm cell strainers, pelleted, and resuspended in the ACK lysis buffer as described in the thymus tissue collection section. Cell density and viability were determined using Countness II Cell Counter. Subsequently, splenocytes were resuspended at 1 million cells per ml of 0.04% BSA/PBS. In order to sort out the splenocyte T-cell subsets for constructing 10× GEM single-cell libraries, splenocytes were resuspended at 1 million cells per ml of FACS buffer (PBS with 1% FBS and 5mM EDTA).splenocytes were pre-incubated with the isotype control antibodies (100 ug/ml mIgG1 & 50 ug/ml rat IgG1) per 1 million cells/100ul FACS buffer for 10 minutes. Following that, cells were washed and resuspended at 2 million cells per 1 ml FACS buffer, and incubated with an anti-NMR CD3e (Abbvie-clone-5, Alexa 647, 1:100) and an anti-mouse CD11b (which cross reacts with the NMR CD11b,) (M1/70, R-PE, 5 ug/ml) on ice for 40 minutes. Cells were then washed and resuspended in the FACS buffer containing Sytox Blue Live/Dead dye for cell sorting using FACS BD Aria. Viable, single cells were gated, and the CD11b-/CD3e+ cells were collected for the generation of 10× GEM single-T-cell libraries.
Single cells were captured in droplet emulsion using the Chromium Controller (10× Genomics, Pleasanton, CA), and GEM single-cell libraries were constructed according to the 10× Genomics protocol using the Chromium Single-Cell 30 Gel Bead and Library 3’ V3 kit (10× Genomics, Pleasanton, CA). In brief, cell suspensions were diluted in PBS with 0.04% BSA to a final concentration of 1×106 cells/mL (1,000 cells per μL). Cells were loaded in each channel with a target output of 10,000 cells per sample. All reactions were performed in a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) with a 96 Deep Well Reaction Module. Twelve cycles were used for cDNA amplification and sample index PCR. Amplified cDNA and final libraries were evaluated using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) with a high sensitivity chip.
paired-end ilimina next-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10x Genomics
nmr_spleen.T.cells_24y_3
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| Data processing |
Cell Ranger mkfastq for demultiplexing
Cell Ranger count for generating gene-barcode-matrix
HetGla2.0 + GCF_000247695.1 and Heterocephalus_glaber_female 1.0 combined annotations
barcodes.tsv.gz is the gunzipped list of sequenced barcodes. genes.tsv.gz is the gunzipped list of sequenced genes. matrix.mtx.gz is the gunzipped compressed sparse gene-barcode-matrix
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| Submission date |
Sep 28, 2022 |
| Last update date |
Nov 30, 2023 |
| Contact name |
Nimrod Daniel Rubinstein |
| E-mail(s) |
[email protected]
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| Phone |
9193082834
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| Organization name |
Calico
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| Street address |
1170 Veterans blvd
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL24046 |
| Series (1) |
| GSE214390 |
The evolution of the naked mole-rat T cells |
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| Relations |
| BioSample |
SAMN31066227 |
| SRA |
SRX17738185 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6605091_nmr_spleen.T.cells_24y_3_barcodes.tsv.gz |
3.1 Mb |
(ftp)(http) |
TSV |
| GSM6605091_nmr_spleen.T.cells_24y_3_genes.tsv.gz |
353.9 Kb |
(ftp)(http) |
TSV |
| GSM6605091_nmr_spleen.T.cells_24y_3_matrix.mtx.gz |
49.1 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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