|
| Status |
Public on May 20, 2022 |
| Title |
T6 - Tumor Trp1 CD4+ |
| Sample type |
SRA |
| |
|
| Source name |
Sorted T cells
|
| Organism |
Mus musculus |
| Characteristics |
tumor or no tumor: Tumor
sorted t cell population: CD4+
treatment: No treatment
strain: RAG1- BW TRP-1 TCR
|
| Treatment protocol |
Cyclophosphamide (CTX) monohydrate mixed in sterile PBS was administered intraperitoneally as a single dose at 250mg/kg once tumors were established (1-2 weeks later). 1 day following CTX injection, 1x10^6 purified TRP1 CD4+ T cells or PMEL CD8+ T cells were intravenously injected into the tail vein in 100ul PBS.
|
| Growth protocol |
B16F10 melanoma cells (2x105 cells) were implanted subcutaneously in 0.2ml of matrigel. Tumors were resected 11 days after T cell transfer and TRP1 (CD45.1) CD4+ and CD4+CD8+, and Pmel (CD90.1) CD8+ and CD8+CD4+ TILS were FACs sorted. Naïve Trp1 CD4+ and Pmel CD8+ T cells were sorted directly from the spleen of naive transgenic mice.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were isolated from 3 independent experiments of pooled spleens (n=3) or tumors (n=5-8) as follows: (i)Naïve TRP1 CD4+ T cells and naïve PMEL CD8+ T cells were sorted by flow cytometry from spleens of Trp1 and Pmel-1 transgenic mice. (ii) TRP1 CD4+ and TRP1 CD4+CD8+ were sorted from B16 tumors of C57BL/6 mice that had been adoptively transferred with naïve TRP1 CD4+ T cells 11 days prior. (iii) PMEL CD8+ and PMEL CD4+CD8+ were sorted from B16 tumors of C57BL/6 mice that had been adoptively transferred with naïve PMEL CD8+ T cells 11 days prior. Cells were gated on congenic labels (TRP1 – CD45.1, PMEL - CD90.1), CD3+, CD4+CD8- (TRP1), CD4+CD8+ (TRP1 and PMEL), or CD4-CD8+ (PMEL). Samples were directly sorted into Trizol (Invitrogen), adjusted to 500uL total volume and frozen and stored at −80 °C. RNA was extracted using RNeasy mini kit (Qiagen) per instructions provided by the manufacturer.
After ribogreen quantification and quality control of Agilent BioAnalyzer, total RNA underwent amplification using the SMART-seq V4 (Clonetech) ultralow input RNA kit for sequencing (12 cycles of amplification for 2–10 ng of total RNA). Subsequently, 10 ng of amplified cDNA was used to prepare Illumina Hiseq libraries with the Kapa DNA library preparation chemistry (Kapa Biosystems). Samples were run on a Hiseq 4000, in a 50-bp/50-bp paired-end run, using the TruSeq SBS Kit v3 (Illumina).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Bulk RNAseq for each sample (FASTQ) were aligned to corresponding genome with STAR (v2.6.0)
Count based expression profiles were quantified using Subread featureCounts (v1.5.1)
Assembly: mm10 (GRCm38.75)
|
| |
|
| Submission date |
May 17, 2022 |
| Last update date |
May 20, 2022 |
| Contact name |
Bic MSKCC |
| Organization name |
Memorial SLoan-Kettering Cancer Center
|
| Street address |
1275 York Ave.
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE203186 |
Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions (4) |
| GSE203190 |
Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions |
|
| Relations |
| BioSample |
SAMN28488931 |
| SRA |
SRX15319661 |
