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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 20, 2022 |
| Title |
DPTCells_RNA_CD4+,CD8+,CD4+CD8+,CD8+CD4+ |
| Sample type |
SRA |
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| Source name |
Sorted T cells from B16 melanoma resections
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| Organism |
Mus musculus |
| Characteristics |
cell type: Tumor infiltrating T cells
congenic marker: CD45.1,CD45.1,CD45.2,CD45.2
treatment: untreated
dna id: C0301,C0302,C0303,C0304
dna barcode: ACCCACCAGTAAGAC,GGTCGAGAGCATTCA,CTTGCCGCATGTCAT,AAAGCATTCTTCAG
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| Treatment protocol |
1 day following tumor implantation, 1x10^6 purified CD45.1 CD4+ or CD45.2 CD8+ T cells were intravenously injected into the tail vein in 100ul PBS.
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| Growth protocol |
B16F10 melanoma cells (2x105 cells) were implanted subcutaneously in 0.2ml of matrigel. Tumors were resected 11 days later and CD45.1 CD4+ and CD4+CD8+, and CD45.2 CD8+ and CD8+CD4+ TILS were FACs sorted.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Intra-tumor CD45.1+ CD4+, CD45.1+ CD4+CD8+, CD45.2+ CD8+, and CD45.2+ CD8+CD4+ T cells were sorted as individual populations from the tumors of 10 pooled mice. Sorted T cells were individually hashtagged and counted. Approximately equal numbers of each cell population were pooled togther as one sample for sequencing. Sorted T cells were stained with Trypan blue and the Countess II Automated Cell Counter (ThermoFisher) was used to assess both cell number and viability. Following QC, the single cell suspension was loaded onto Chromium Next GEM Chip K (10X Genomics PN 1000286) and GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 6,200 cells proceeded using the Chromium Next GEM Single Cell 5’ Kit v2 (10X Genomics PN 1000263) according to the manufacturer’s protocol. cDNA amplification included 14 cycles and 10.6ng of the material was used to prepare a sequencing library with 16 cycles of PCR. The indexed library was pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/91 run using the NovaSeq 6000 SP Reagent Kit (100 cycles) (Illumina). 194 million paired reads were generated for the sample.
An aliquot of cDNA generated using the methods described above was used to enrich for V(D)J regions using the Chromium Single Cell Mouse TCR Amplification Kit (10X Genomics PN 1000254) according to the manufacturer’s protocol with 10 cycles of PCR during enrichment and 9 cycles during library preparation. The indexed library was pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/91 run using the NovaSeq 6000 SP Reagent Kit (100 cycles) (Illumina). 39 million paired reads were generated for the sample. Amplification products generated using the methods described above included both cDNA and feature barcodes tagged with cell barcodes and unique molecular identifiers. Smaller feature barcode fragments were separated from longer amplified cDNA using a 0.6X cleanup using aMPure XP beads (Beckman Coulter catalog # A63882). Libraries were constructed using the 5’ Feature Barcode Kit (10X Genomics PN 1000256) according to the manufacturer’s protocol with 9 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/91 run using the NovaSeq 6000 SP Reagent Kit (100 cycles) (Illumina). 72 million paired reads were generated for the sample.
scRNA/TCR sequencing with hasthtag and feature barcoding
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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 scRNA
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| Data processing |
Filtered gene expression matrices were generated using 10X CellRanger (version 3.1.0).
Paired single-cell VDJ sequences and annotations were generated using 10X Cellranger VDJ (version 3.1.0) and high confidence sequences were mapped to gene expression barcodes.
Feature barcode matrices were generated for each sample (consisting of five mice) using antibody barcode patterns from TotalSeq.
Mouse specific barcodes were demultiplexed by HTO using Seurat (version 3.2.3).
Assembly: mm10 (GENCODE vM23/Ensembl 98)
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| Submission date |
May 17, 2022 |
| Last update date |
May 20, 2022 |
| Contact name |
Bic MSKCC |
| Organization name |
Memorial SLoan-Kettering Cancer Center
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| Street address |
1275 York Ave.
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE203184 |
Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions (3) |
| GSE203190 |
Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions |
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| Relations |
| BioSample |
SAMN28488851 |
| SRA |
SRX15303416 |
| 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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