|
| Status |
Public on May 20, 2022 |
| Title |
T2 - ATAC-Seq - Naïve Trp1 CD4+ |
| Sample type |
SRA |
| |
|
| Source name |
Sorted T cells
|
| Organism |
Mus musculus |
| Characteristics |
tumor or no tumor: No 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 |
genomic DNA |
| 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. After flow-sorting, all samples for downstream ATAC-seq analysis were frozen in 10% DMSO and stored at −80 °C.
Profiling of chromatin was performed by ATAC-seq as described by Buenrostro, et al34. Briefly, 8-50,000 viably frozen T cells were thawed, washed in cold PBS, and lysed. The transposition reaction containing TDE1 Tagment DNA Enzyme (Illumina catalog # 20034198) was incubated at 37°C for 30 minutes. The DNA was cleaned with the MinElute PCR Purification Kit (QIAGEN catalog # 28004) and material was amplified for 5 cycles using NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs catalog # M0541L). After evaluation by real-time PCR, 7-13 additional PCR cycles were done. The final product was cleaned by aMPure XP beads (Beckman Coulter catalog # A63882) at a 1X ratio, and size selection was performed at a 0.5X ratio. Libraries were sequenced on a HiSeq4000 in a PE50 run, using the HiSeq 3000/4000 SBS Kit (Illumina). An average of 57 million paired reads were generated per sample.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
ATAC-seq data were aligned to the mm9 reference genome. Alignment and peak calling were performed by atac-seq-pipeline v1.7.1 from ENCODE Data Coordination Center (ENCODE DCC).
|
| |
|
| 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) |
| GSE203188 |
Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions (6) |
| GSE203190 |
Tumor-induced double positive T cells display distinct lineage commitment mechanisms and functions |
|
| Relations |
| BioSample |
SAMN28488953 |
| SRA |
SRX15319529 |
