characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-06-30T00:24:29Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-06-30T00:24:29Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA113611590
characteristics.characteristic: External Id
characteristics.text: Genentech
characteristics.characteristic: INSDC center name
characteristics.text: 2023-06-30T00:24:29Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-06-30T00:24:29Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS15605769
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-13079:aTGFb_3
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: T cell
characteristics.characteristic: cell_type
characteristics.text: not collected
characteristics.characteristic: collection date
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: Tumors were collected 7 days after treatment initiation. Tumors were weighed and enzymatically digested using a cocktail of dispase, collagenase P, and DNaseI for 45 min at 37°C, to obtain a single-cell suspension. Cells were counted using a Vi-CELL XR (Beckman Coulter, Brea, CA). Cells were first enriched for live cells using Dead Cell Removal Kit following the manufacturer’s instructions. Cells were then incubated with mouse BD Fc block (5 µg/ml) for 30 min on ice. And then stained for 30 minutes on ice with the following antibodies: CD45 PE-Cy7 (1 µg/ml), TCR-β APC (1 µg/ml), THY1.1 AF488 (2.5 µg/ml), PDPN PE (0.5 µg/ml), CD31 BUV737 (1 μg/ml), CD11b BUV395 (1 μg/ml). Just before sorting cells were stained with 7AAD and eBioscience™ Calcein Blue AM Viability Dye. Four population were sorted: T cell (7AAD- Calcein blue+ CD45+ THY1.1- TCRb+), fibroblasts (7AAD- Calcein Blue+ CD45- THY1.1- CD31- PDPN+), myeloid cells (7AAD- Calcein Blue+ CD45+ THY1.1- CD11b+) and tumor cells (7AAD- Calcein Blue+ CD45- THY1.1+). Cells were sorted using a BD FACSAria™ Fusion flow cytometer (BD Biosciences, San Jose, CA). 5k cells for each population per mouse were sorted in 100 UL of RA1 buffer + 2 UL of TCEP (NucleoSpin RNA XS) spun down, and immediately frozen in dry ice. Samples were conserved at -80C until RNA extraction. RNA was isolated using NucleoSpin RNA XS isolation kit (TAKARA Bio USA Inc.) following manufacturer’s instruction. cDNA library was generated from 2 nanograms of total RNA using Smart-Seq V4 Ultra Low Input RNA Kit (Takara). 150 picograms of cDNA were used to make sequencing libraries by Nextera XT DNA Sample Preparation Kit (Illumina). Libraries were quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the average library size was determined using D1000 ScreenTape on TapeStation 4200 (Agilent Technologies).
characteristics.characteristic: description
characteristics.text: not collected
characteristics.characteristic: geographic location (country and/or sea)
characteristics.text: 7AAD- Calcein blue+ CD45+ THY1.1- TCRb+
characteristics.characteristic: immunophenotype
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: tumor
characteristics.characteristic: sampling site
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: aTGFb_3
characteristics.characteristic: title