 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 12, 2024 |
| Title |
#1 memory CD8+ T cells, blood, 4 hour stimulation |
| Sample type |
SRA |
| |
|
| Source name |
peripheral blood
|
| Organism |
Homo sapiens |
| Characteristics |
donor: #1
serotype: CMV positiv
tissue: peripheral blood
cell type: CD8 T cell
marker: CD3+/CD4-/CD8+/CCR7-/CD28_high/CD27+/CD45RA-
treatment: 4 hour stimulation with CMVpp65 overlapping peptide pool
|
| Treatment protocol |
Pre-enriched human CD8+ Tcells were stimulated with CMVpp65 overlapping peptide pool (Miltenyi Biotec) at a concentration of 1 µg of each peptide/ml for 4 hours in TexMACS medium (Miltenyi Biotec).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
PBMCs were isolated from residual blood samples from disposable kits used during routine platelet apheresis via discontinuous gradient centrifugation. CD8+ T cells were pre enriched using human anti-CD8 MicroBeads and the automated magnetic activated cell sorting (autoMACS) system (Miltenyi Biotec) according to the manufacturer’s instructions. The cells were stimulated with CMVpp65 overlapping peptide pool (Miltenyi Biotec) at a concentration of 1 µg of each peptide/ml for 4 hours. Subsequently, IFN γ secreting cells were labelled using IFN γ Secretion Assay/Detection Kit (PE) (Miltenyi Biotec; manufacturer’s protocol) and antibodies for surface protein detection. Genomic DNA was prepared from flow cytometrically sorted naive CD8+ T cells, interferon-gamma negative memory CD8+ T cells and interferon-gamma positive CD8+ T cells by using DNeasy blood and tissue kit (Qiagen, manufacturer’s protocol). Genomic DNA was bisulfite converted using the EZ DNA Methylation Kit (Zymo Research, manufacturer’s protocol) and subjected to library preparation.
The bisulfite converted DNA was fragmented by sonication and served as input for library preparation using the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences, manufacturer’s protocol).
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Illumina RTA3 was used for basecalling.
Sequencing data was processed using the nf-core/methylseq pipeline (version 2.2.0), using default parameters, genome assembly GRCh38 and --accel=true (DOI:10.5281/zenodo.1343417, DOI:10.1038/s41587-020-0439-x). Briefly, the pipeline employs FASTQC (version 0.11.9), trimgalore (version 0.6.7) and bismark (version 0.24.0) (DOI:10.1093/bioinformatics/btr167) for read-level quality control, adapter trimming, bisulfite-aware alignment and cytosine-level DNA methylation quantification.
Assembly: GRCh38
Supplementary files format and content: *.bismark.cov.gz files contain CpG-level methylation calls in bismark covareage format
|
| |
|
| Submission date |
Oct 19, 2023 |
| Last update date |
Sep 12, 2024 |
| Contact name |
Fabian Müller |
| Organization name |
Saarland University
|
| Street address |
Campus A 2.4
|
| City |
Saarbrücken |
| ZIP/Postal code |
66123 |
| Country |
Germany |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE245832 |
DNA methylation profiling identifies TBKBP1 as potent amplifier of cytotoxic activity in CMV-specific human CD8+ T cells |
|
| Relations |
| BioSample |
SAMN37887192 |
| SRA |
SRX22149901 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7849754_Tmem_D1.bismark.cov.gz |
205.2 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
