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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 02, 2023 |
| Title |
OVAtetramer_low |
| Sample type |
SRA |
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| Source name |
Spleen
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| Organism |
Mus musculus |
| Characteristics |
tissue: Spleen
cell line: primary T cell
cell type: CD8+ T cells (OVA-tetramer-low)
genotype: WT
treatment: Listeria infection
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| Treatment protocol |
CRISPR/Cas9 technology was used to generate NGF knockout stable cell lines. C57BL/6 mice were vaccinated i.v. with 0.1 LD50 of attenuated recombinant Listeria-OVA strain.
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| Growth protocol |
For B16OVA library, B16OVA cells were subcutaneously injected into C57BL/6J mice. Total RNA was extracted from melanoma tissues day 11 and analyzed by RNA-seq. For YUMM library, YUMM1.7 cells were subcutaneously injected into C57BL/6J mice. Total RNA was extracted from melanoma tissues day 12 and analyzed by RNA-seq. For dLN_KO library, C57BL/6J mice were subcutaneously injected with 50,000 Ctrl or NGF sgRNA B16-OVA cells. All mice in the Ctrl sgRNA group were sacrificed when tumor growth limits were exceeded. Tumor-free mice in the NGF sgRNA group were rechallenged with B16-OVA cells on day 78. 9 days later, OVA-tetramer+ CD8+ T cells were sorted from tumor draining lymph nodes and submitted for TCRb-seq. For dLN_NT_aPD-1 and dLN_KO_aPD-1 library, C57BL/6J mice were subcutaneously injected with 50,000 Ctrl or NGF sgRNA B16-OVA cells. 250 ug/mouse anti-PD-1 antibody was given twice. Tumor-free mice in both groups were rechallenged with B16-OVA cells on day 78. 9 days later, OVA-tetramer+ CD8+ T cells were sorted from tumor draining lymph nodes and submitted for TCRb-seq. For OVAtetramer_high and OVAtetramer_low library, C57BL/6 mice were vaccinated i.v. with 0.1 LD50 of attenuated recombinant Listeria-OVA strain. OVA-tetramer-high and –low CD8 T cells were FACS-sorted from spleen on day 7 after a single vaccination, and then submitted for TCRb-seq.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from OVA-tetramer+ CD8+ T cells using an RNeasy Mini Kit (Invitrogen) or from bulk tumor tissues using a Direct-zol RNA kit (ZYMO Research).
1 μg or lower amounts of RNA were converted into cDNA using a cDNA Synthesis Kit (Quantabio) with a constant region-specific primer. A multiplex PCR system was introduced to amplify the CDR3 region of rearranged TCRB loci. A set of forward primers, each specific to one or a set of functional TCR Vb segments, and a reverse primer specific to the constant region of TCRB, were used to generate amplicons covering the entire CDR3 region. PCR products were loaded onto 2.5% agarose gels, and bands centered at 250–400 bp were excised and purified using a QIAquick Gel Extraction kit (QIAGEN).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
TCRseq data was assembled and aligned to reference V, D, J, and C genes of T-cell receptors using MiXCR v2.19 and built-in reference database. Specifically, a two-round assembly, including a first-round partial assembly and a second-round full assembly, was conducted to capture more clonotypes, referred to a specific CDR3 sequence. High-quality reads were firstly assembled into core clonotypes, which were then extended based on unique V and J genes against germline sequences. The final assembly was performed with error correction algorithms to merge similar clonotypes and their abundance.
Supplementary files format and content: clonotypes and their abundance
Library strategy: TCR-seq
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| Submission date |
Sep 22, 2023 |
| Last update date |
Nov 02, 2023 |
| Contact name |
Liuyang Wang |
| Organization name |
Duke University
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| Street address |
213 Research Drive
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| City |
Durham |
| State/province |
NC |
| ZIP/Postal code |
27710 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE236682 |
Breaking through NGF-TrkA immunosuppression in melanoma sensitizes immunotherapy for durable memory T cell protection |
| GSE243814 |
Breaking through NGF-TrkA immunosuppression in melanoma sensitizes immunotherapy for durable memory T cell protection [TCR-seq] |
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| Relations |
| BioSample |
SAMN37516063 |
| SRA |
SRX21860336 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7796003_OVAtetramer_low_CKDL220013124-1B_HHM2HBBXX_TRB.txt.gz |
3.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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