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Sample GSM7957349 Query DataSets for GSM7957349
Status Public on Sep 11, 2024
Title CD4_CD8_NKT_MAIT_GD_Thymus_D
Sample type SRA
 
Source name Thymus
Organism Homo sapiens
Characteristics tissue: Thymus
Sex: M
age: 10 weeks
Extracted molecule polyA RNA
Extraction protocol To extract thymocytes for both single-cell RNA sequencing (scRNAseq) and flow cytometry, the thymus tissue was placed in complete RPMI 1640 media (Gibco, #22400-071) (10% heat-inactivated fetal bovine serum (FBS, Sigma-Aldrich), 1% non-essential amino acids (Sigma-Aldrich), 1% Sodium Pyruvate (Sigma-Aldrich), 1X GlutaMAX (Gibco), 1% Penicillin/Streptomycin (Gibco), and 1X 2-mercaptoethanol (BME, Sigma-Aldrich)), cut into small pieces, and gently pressed with the back of a 10 ml syringe to release thymocytes. The resulting suspension was passed through a 70 µm filter. Thymocytes and PBMCs were isolated using a Ficoll-Paque density gradient provided by Cytiva. PBMCs were cryopreserved in FBS with 10% DMSO from Sigma-Aldrich and stored in liquid nitrogen. Tetramer staining for MAIT and iNKT cells was performed on freshly isolated thymocytes. For tetramer staining of MAIT and iNKT cells, freshly isolated thymocytes were used. To enrich for thymic MAIT and thymic/blood iNKT cells, up to 2x109 cells were incubated with MR1-5-OP-RU-PE-Tet or CD1d-PBS57-PE respectively in MACS buffer (0.5% BSA, 2mM ETDA, PBS), for 25 mins at room temperature. Cells were washed twice and incubated with anti-PE microbeads (Miltenyi), followed by separation using an autoMACS Pro Separator (Miltenyi) according to manufacturer’s instructions. Enriched MAIT and iNKT from thymus, enriched iNKT from PBMC, unenriched gd T ,CD4+ and CD8+ from thymus, and unenriched MAIT, gd T , CD4+ and CD8+ T cells from PBMC were stained with the following cell surface markers in MACS buffer at room temperature for 20 mins: CD3-AF488 (clone OKT3, Biolegend), CD14-eFluor450 (clone 61D3, ThermoFisher), CD19-eFluor450 (clone H1B19, ThermoFisher), Va7.2-BV785 (clone 3C10, Biolegend), Va24-PerCP-Cy5.5 (clone C15, Biolegend), CD4-AF710 (clone OKT4, Tonbo), CD8a-PE-Cy7 (clone SK1, Tonbo), TCR-BV650 (clone 11F2, BD Biosciences), FcgR block (Miltenyi). Cells were washed twice and resuspended in MACS buffer prior to cell sorting on the Aria 3 (BD Biosciences).
Single cell whole transcriptomes and TCR sequencing libraries were prepared using the BD Rhapsody Single-Cell Analysis System (BD Biosciences) according to the manufacturer's specifications. Prior to cell sorting on the Aria 3 (BD Biosciences) and during cell surface antibody staining, up to 2 x106 enriched or unenriched cells were labeled with an oligonucleotide-tagged antibody sample tag (BD Biosciences). From infant thymus and PBMC donors up to 5 populations were sorted after doublet, viability, B cell (CD19+CD3-) and monocyte (CD14+CD3-) discrimination: 1. MAIT cells (MR1-5-OP-RU-Tet+Va7.2+CD3+), 2. iNKT cells (CD1d-PBS57-Tet+Va24+CD3+), 3. gd T cells (CD3+TCRgd+), 4. CD4+ T cells (CD4+CD8-CD3+) and CD8+ T cells (CD8+CD4-CD3+). Prior to cDNA library preparation for the WTA and VDJ libraries, all cell subsets from the different donors were pooled, with up to 12 unique sample tags combined per library. Libraries were quantified and pooled according to equivalent molar concentrations and sequenced on the NovaSeq sequencing platform at the University of Colorado Genomics Core with the following read lengths: read 1 – 150 cycles; read 2 – 150 cycles; and i7 index - 8 cycles.
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description BD Rhapsody
Data processing The demultiplexing, barcoded processing, gene counting and aggregation were made by the BD Rhapsody WTA Analysis Pipeline software (revision 4)
Assembly: GRCh38
Supplementary files format and content: comma-separated raw count matrix
Supplementary files format and content: comma-separated VDJ
 
Submission date Dec 08, 2023
Last update date Sep 11, 2024
Contact name Tonya Brunetti
E-mail(s) tonya.brunetti@cuanschutz.edu
Organization name University of Colorado Anschutz Medical Campus
Department Immunology and Microbiology
Street address 12800 East 19th Avenue
City Aurora
State/province CO
ZIP/Postal code 80045
Country USA
 
Platform ID GPL24676
Series (1)
GSE249684 Unraveling the Phenotypic States of Human innate-like T Cells in thymus and blood: Insights from Single-Cell Transcriptomics
Relations
BioSample SAMN38727357
SRA SRX22828820

Supplementary file Size Download File type/resource
GSM7957349_CUThy13_220225_SampleTag01_hs_CD4_RSEC_MolsPerCell.csv.gz 2.7 Mb (ftp)(http) CSV
GSM7957349_CUThy13_220225_SampleTag02_hs_CD8_RSEC_MolsPerCell.csv.gz 2.5 Mb (ftp)(http) CSV
GSM7957349_CUThy13_220225_SampleTag03_hs_GD_RSEC_MolsPerCell.csv.gz 5.2 Mb (ftp)(http) CSV
GSM7957349_CUThy13_220225_SampleTag04_hs_MAIT_RSEC_MolsPerCell.csv.gz 1.5 Mb (ftp)(http) CSV
GSM7957349_CUThy13_220225_SampleTag05_hs_NKT_RSEC_MolsPerCell.csv.gz 999.2 Kb (ftp)(http) CSV
GSM7957349_CUThy13_220225_VDJ_Dominant_Contigs.csv.gz 3.0 Mb (ftp)(http) CSV
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Raw data are available in SRA

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