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| Status |
Public on Aug 05, 2020 |
| Title |
blood_Tcm_6 |
| Sample type |
SRA |
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| Source name |
blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
cell type: CLA+CD4+ T cells
cell population: CM
acquisition site: Salzburg
donor: 6
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Human PBMCs were isolated using Ficoll-Hypaque (GE-Healthcare; GE17-1440-02) gradient separation. For RNAseq CD4+ T cells were enriched using CD4 microbeads (Miltenyi; 130-045-101) and 4x106 cells/ml were resuspended in RPMIc with 50 U/ml IL-2 (Immunotools; 11340023) in a 24-well and rested for 20h prior to staining and cell sorting. For isolation of skin T cells, subcutaneous fat was removed before skin was minced with dissection scissors and surgical scalpel. Approximately 1cm^2 of skin was digested overnight in 5%CO2 at 37°C with 3ml of digestion mix containing 0.8mg/ml Collagenase Type 4 (Worthington; #LS004186) and 0.02mg/ml DNase (Sigma-Aldrich; DN25) in RPMIc. Samples were filtered, washed with RPMI and PBS and stained for flow cytometry/cell sorting.
500 cells per population were sorted into lysis buffer using a BD FACSARIA III or FACSFUSION instruments (BD Biosciences), and cDNA was prepared using the SMARTSeq v4 Ultra Low Input RNA Kit for Sequencing (Takara).
Library construction was performed using the NexteraXT DNA sample preparation kit (Illumina) using half the recommended volumes and reagents.
sequencing run: Dual-index, single-read sequencing of pooled libraries was run on a HiSeq2500 sequencer (Illumina) with 58-base reads and a target depth of 5 million reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
lib16882
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| Data processing |
Base-calling and demultiplexing were performed automatically on BaseSpace (Illumina) to generate FASTQ files.
The FASTQ files were processed in order to remove reads of zero length (fastq_trimmer v.1.0.0), remove adapter sequences (fastqmcf tool v.1.1.2) and perform quality trimming from both ends until a minimum base quality ≥ 30 (FASTQ quality trimmer tool v.1.0.0).
Reads were aligned to the human reference genome (build hg38) with TopHat (v.1.4.0) and read counts per Ensembl gene ID were quantified with htseq-count (v.0.4.1).
Quality metrics for the FASTQ and BAM/SAM files were generated with FastQC (v.0.11.3) and Picard (v.1.128).
Processing of FASTQ and BAM/SAM files was executed on the Galaxy workflow platform of Globus genomics.
Samples with a total number of fastq reads below 10^6, mapped reads below 70% or median CV coverage > 1 were excluded from further analysis.
gene_counts.txt: Gene counts as determined by htseq-count. Ensembl Gene IDs were mapped to HGNC gene symbols using biomaRt (GRCh38.p10). Genes with low counts as well as non-protein coding genes were removed for downstream analyses.
Genome_build: hg38
Supplementary_files_format_and_content: gene_counts.txt
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| Submission date |
Apr 20, 2020 |
| Last update date |
Aug 05, 2020 |
| Contact name |
Daniel J Campbell |
| Organization name |
Benaroya Research Institute
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| Street address |
1201 Ninth Avenue
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98101 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE148970 |
Transcriptional analysis of resting T cells from human blood and skin |
| GSE149090 |
Transcriptomic profiling of human effector and regulatory T cell subsets identifies predictive population signatures |
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| Relations |
| BioSample |
SAMN14646314 |
| SRA |
SRX8143624 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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