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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 19, 2024 |
| Title |
PAM007, CD8 T cells, scRNAseq |
| Sample type |
SRA |
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| Source name |
Endometrial tumor tissue
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Endometrial tumor tissue
cell type: CD8 T cell
genotype: MMRd tumor, WT T cells
treatment: Pembrolizumab 200mg Q3W; 2 cycles
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| Treatment protocol |
Patients were treated with neoadjuvant pembrolizumab Q3W for 2 cycles.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tumour digests were thawed in FCS and resuspended in RPMI + 10% FCS. After 1.5h incubation at 37°C/5% CO2, samples were stained and incubated with a CXCR5 PerCP-Cy5.5 antibody for an additional 0.5h at 37°C. After a total of 2h recovery at 37°C/5% CO2, samples were centrifuged and resuspended in 100 µl PBS + 2% FCS. Samples were incubated for 30-45 minutes on ice (in dark) with the following antibodies: CD45 BV605, CD8 APC Cy7, CD4 PE, CD19 BV421, PD1 APC, CD3 PE-Cy7 and the dump channel antibodies CD14 FITC, CD56 FITC and EpCAM FITC. 3 µl of each antibody was used per 1x10e6 cells. Samples were washed twice with PBS + 2% FCS and filtered using a 35 µm strainer (Falcon) before sorting on a Beckman Coulter MoFlo Astrios cytometer. UltraComp eBeads (Thermo Fisher Scientific) were used as compensation controls. Before sorting, Propidium Iodide was added to exclude dead cells. Sorting was done in 1.5 mL Low Bind DNA tubes (Eppendorf) in 150 µl PBS 0.04 % BSA. CD45+ CD3+ CD19- CD8+ and CD4+ cells were sorted. Sorted cells were centrifuged and resuspended at a concentration of 727 cells/µl (total 16.5 µl) for PAM-007 or 2121 cells/µl (total 16.5 µl) for PAM-010 and PAM-011, in PBS 0.04% BSA.
Library was performed according to manufacturer’s protocol (10x genomics): ‘chromium next GEM single cell 5’ reagents kit v2 (dual index)’. For the cDNA amplification step, 13 cycli were done. For each sample, both the V(D)J library as well as the gene expression (GEX) library was constructed. All cDNA quality control and quantification steps were done with the Qubit 4 Fluorometer (Thermo Fisher) and the Agilent 4200 Tapestation (with D5000 and high sensitivity D5000 screen tapes). For the sequencing, V(D)J and GEX libraries were pooled at a 1:4 molar ratio, with 2 GEX libraries and 2 V(D)J libraries in one pool. 1.5 pM of this superpool was sequenced on a NextSeq 500 (Illumina inc., San Diego, CA, USA) A 150 bp NextSeq high-output reagent kit was used for the sequencing (paired end).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Description |
10x Genomics
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| Data processing |
Reads from single cells isolated using 10x chromium were demultiplexed and aligned to the GRCh38.p12 human reference genome (from 10x Genomics) using Cell Ranger (version 6.0.1; 10x Genomics)).
Assembly: GRCh38.p12
Supplementary files format and content: Tab-separated values files, matrix files and comma-separated values files
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| Submission date |
Jun 03, 2024 |
| Last update date |
Jul 19, 2024 |
| Contact name |
Marco de Bruyn |
| E-mail(s) |
m.de.bruyn@umcg.nl
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| Organization name |
University Medical Center Groningen
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| Department |
Obstetrics&Gynecology
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| Lab |
Immuno-Oncology
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| Street address |
Hanzeplein 1
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| City |
Groningen |
| ZIP/Postal code |
9713 GZ |
| Country |
Netherlands |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE268903 |
Neoadjuvant immune checkpoint blockade in women with mismatch repair deficient endometrial cancer: a phase I study |
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| Relations |
| BioSample |
SAMN41656138 |
| SRA |
SRX24781430 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8302395_PAM007_CD8_barcodes.tsv.gz |
19.8 Kb |
(ftp)(http) |
TSV |
| GSM8302395_PAM007_CD8_features.tsv.gz |
325.6 Kb |
(ftp)(http) |
TSV |
| GSM8302395_PAM007_CD8_matrix.mtx.gz |
24.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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