title: Tumor_SPF_GEX
organism: Mus musculus
description: Protocols: CFSE+ cells were collected from tumor, tumor draining lymph node (tdLN), contralateral lymph node (cLN) and mesenteric lymph nodes (mLN), 24H after CFSE injection in mLN, in MCA205 tumor-bearing mice, colonized (E.clostri) or not (SPF) by Enterocloster clostridioformis bacteria. CFSE+ CD4 and CD8 T cells were collected with cell sorter (cytometry). Tumors, mesenteric lymph nodes (mLN), draining lymph nodes (tdLN) and contralateral lymph nodes (cLN) were harvested 24h after CFSE injection in the mLN, 10 days after tumor inoculation. Excised tumors were cut into small pieces and digested in RPMI medium containing LiberaseTM at 25 μg/ml (Roche) and DNase1 at 150 IU/ml (Roche) for 30 minutes at 37°C and then crushed and filtered twice using 100- and 70-μm cell strainers (Becton & Dickinson). Lymph nodes were crushed in RPMI medium and subsequently filtered twice through a 100-μm cell strainer. MCA205 were inoculated subcutaneously at a concentration of 0.8x10^6 cells/mL in 100µl. Tumor growth lasted 10 days. Mice were oral with water or E. clostridioformis at day 5 and day 7 after tumor inoculation. Single cell suspensions from a total of 500 to 20,000 cells with barcoded gel beads and partitioning oil were loaded to Chromium Next GEM Chip K to generate single-cell gel bead-in-emulsion. Full-length cDNA along with cell barcode identifiers were PCR-amplified to generate 5' Gene Expression (GEX) libraries and V(D)J libraries. Libraries construction were performed using Chromium Next GEM Single Cell 5' kits v2 (PN-1000263), Chromium Next GEM Chip K, Single Cell VDJ 5' Gel Bead, Chromium Single Cell Mouse TCR amplification kit (16 rxns (reactions)) PN-1000254, Library construction kit (16 rxns) PN-1000190 and Dual Index kit TT set A (96 rxns) PN-1000215 kits according to the manufacturer's protocol.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2023-07-01T00:20:36Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-07-01T00:20:36Z
attributes.tag: External Id
attributes.value: SAMEA112898441
attributes.tag: INSDC center alias
attributes.value: Gustave Roussy Cancer Campus, Villejuif Cedex, France
attributes.tag: INSDC center name
attributes.value: Gustave Roussy Cancer Campus, Villejuif Cedex, France
attributes.tag: INSDC first public
attributes.value: 2023-07-01T00:20:36Z
attributes.tag: INSDC last update
attributes.value: 2023-07-01T00:20:36Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-12846:Tumor_SPF_GEX
attributes.tag: age
attributes.value: 6 to 7
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_type
attributes.value: T cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: fibrosarcoma
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: lymph node
attributes.tag: sample name
attributes.value: E-MTAB-12846:Tumor_SPF_GEX
attributes.tag: sampling site
attributes.value: neoplasm
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/6J
attributes.tag: xenograft
attributes.value: MCA205 fibrosarcoma (0.8x10^6 cells) incoculated subcutaneously