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| Status |
Public on Jun 09, 2023 |
| Title |
WT_Tum_2_GEX |
| Sample type |
SRA |
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| Source name |
MC38-OVA colon carcinoma
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| Organism |
Mus musculus |
| Characteristics |
tissue: MC38-OVA colon carcinoma
cell type: CD45+ leukocytes
genotype: WT
treatment: Inplanted MC38-OVA tumors
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| Treatment protocol |
MC38-OVA (0.5x10^6) cells were subcutaneously injected into the right flank of mice.
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| Growth protocol |
C57BL/6J wildtype and B6.129S4-Pglyrp1tm1.1Lky/J mice were obtained from the Jackson Laboratory.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For the isolation of TILs, tumor tissue was dissociated with collagenase D (2.5mg/ml) at 37 degrees Celsius for 20 min, followed by centrifugation through a discontinuous Percoll gradient (GE Healthcare). 1,000 living CD8+ T cells (CD45+ TCRß+ CD8) were FACS-sorted immediately into TCL buffer (QIAGEN) supplemented with 1% ß-mercaptoethanol (Sigma Aldrich). To obtain cells from draining lymph nodes, lymph nodes were mashed through a 40 µm strainer. In the EAE experiment, mice (8-12 weeks old) sex-matched were immunized subcutaneously into the flanks with an emulsion containing the MOG35-55 peptide (100µg/mouse, Genemed Synthesis) and M. tuberculosis H37Ra extract (5mg/ml, Becton Dickinson) in CFA (200ul/mouse, Becton Dickinson). Pertussis toxin (100ng/mouse, List Biological Laboratories) was administered intravenously on days 0 and 2. For the isolation of lymphocytes from EAE mice, mice were sacrificed, and perfusion was performed intracardially with PBS. To obtain cells from the CNS, the forebrain and cerebellum were dissected and together with the spinal cord were flushed out with PBS using hydrostatic pressure. CNS tissue was cut into small pieces with a razor blade and digested with collagenase D (2.5mg/ml, Roche Diagnostics) at 37 degrees Celsius for 20 min. Mononuclear cells were isolated by passing the tissue through a 40 µm strainer, followed by centrifugation through a Percoll gradient (37% and 70%). Lastly, the mononuclear cells were obtained from the interphase, washed, and used for further analysis.
The cell populations of interest were sorted by FACS for sequencing. For cell hashing, the cells were incubated for 30 min on ice in flow buffer with the TotalSeqTM-antibody pool (Biolegend) at 1:1000 final dilution for each antibody, followed by a total of three washes with flow buffer. Following the manufacturer’s instructions (10x Genomics), the samples were then separated into droplet emulsions using the Chromium Single Cell 5' V2 Solution. The library preparations of the scRNA-seq, including TCR-seq and 5' feature barcoding libraries were performed according to manufacturer’s instructions (10x Genomics) using the NextGem 5' v2 Dual Index protocol. Libraries were pooled to 4nM.
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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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| Data processing |
Base-calling was performed using NovaSeq Control Software v1.7.5 (RTA v3.4.4).
The cellrange_workflow in Cumulus (https://cumulus.readthedocs.io/en/stable/cellranger/index.html) was used to preprocess the data. Specifically, 10x Genomics CellRanger 6.0.1 was for BCL to fastq demultiplexing, read alignment (mm10 reference genome for RNA-seq reads and cellranger GRCm38_vdj_v5.0.0 for TCR-seq reads), RNA and hashtag UMI count matrix computation, TCR config identification, and empty droplets filtering.
Cell quality control, doublet detection, tissue origin correction and cell annotation were performed as described in the manuscript. Cleaned and annotated data are available as SingleCellExperiment R objects.
Assembly: mm10
Supplementary files format and content: tumor_rna_cleaned.rds holds a Seurat R object containing RNA-seq expression as a matrix in the RNA assay, cell hashtag data as a matrix in the HTO assay, and cell annotation (including TCR information) in meta.data.
Supplementary files format and content: eae_rna_cleaned.rds holds a Seurat R object containing RNA-seq expression as a matrix in the RNA assay, cell hashtag data as a matrix in the HTO assay, and cell annotation (including TCR information) in meta.data.
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| Submission date |
Jan 27, 2023 |
| Last update date |
Jun 09, 2023 |
| Contact name |
Linglin Huang |
| E-mail(s) |
lhuang@bwh.harvard.edu
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| Organization name |
Brigham and Women’s Hospital
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| Department |
Neurology
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| Lab |
Kuchroo Lab
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| Street address |
60 Fenwood Road, HBTM 10016F
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE223873 |
ScRNA-seq of immune cells in tumors or during EAE from Pglyrp1 KO and wildtype mice. |
| GSE223896 |
Targeting PGLYRP1 promotes anti-tumor immunity while inhibiting autoimmunity |
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| Relations |
| BioSample |
SAMN32941591 |
| SRA |
SRX19200780 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6998220_WT_Tum_2_GEX_filtered_feature_bc_matrix_barcodes.tsv.gz |
8.5 Kb |
(ftp)(http) |
TSV |
| GSM6998220_WT_Tum_2_GEX_filtered_feature_bc_matrix_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM6998220_WT_Tum_2_GEX_filtered_feature_bc_matrix_matrix.mtx.gz |
17.0 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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