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| Status |
Public on Jul 06, 2021 |
| Title |
RNA-seq human CD8+ T IL7+IL15 24h donor1 |
| Sample type |
SRA |
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| Source name |
PBMC
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| Organism |
Homo sapiens |
| Characteristics |
strain: Healthy donors
antibody: CD8+ T cells
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| Treatment protocol |
Purified cells were activated using plate-bound anti-CD3 (2 mg/ml) plus soluble anti-CD28 (1 mg/ml) monoclonal antibodies for two days. After resting overnight in the absence of cytokine, cells were washed and cultured with 1 mg/ml MSA conjugated IL-2, H9 or H9T (unless otherwise specified).
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| Growth protocol |
CD8+ T lymphocytes or draining lymph nodes were isolated using the EasySep Mouse CD8+ T cell isolation kit (STEMCELL #19853) and cultured in RPMI-1640 medium (ATCC) supplemented with 10% FBS and 50 mM b-mercaptoethanol. For RNA-seq using cells isolated from lymph nodes, CD90.1+ CD8 + cells were sorted and cell libraries were prepared with SMART-Seq Ultra low Input RNA kit (Takara, #634889). For human CD8+ T cells related assays, naïve CD8+ T cells were isolated with EasySep™ Human Naïve CD8+ T Cell Isolation Kit II (STEMCELL, #17968), and activated with Dynabeads Human T-Activator CD3/CD28 (Thermo Fisher Scientific, #111.31D) for two days, and cells were isolated by a magnet per the manufacturer’s protocol.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For ChIP-seq, DNA libraries were prepared with the KAPA LTP Library Preparation Kit and barcoded with NEXTflex DNA barcodes. For ATAC-seq, Nuclei were then used for the transposition reaction with the Nextera DNA Library Preparation kit at 37C for 30 min. DNA was purified with the Zymo DNA recovery kit. For RNA-seq, RNA was extracted using the Zymo RNA miniprep kit (Zymo Research), and 500 ng RNA was used for library preparation with the Kapa mRNA HyperPrep Kit (KK8580, Kapa Biosystems) and indexed with NEXTflex DNA Barcodes-24. After the final amplification, samples were loaded onto 2% E-Gel pre-cast gels (ThermoFisher), and 250 to 400 bp DNA fragments were recovered and purified with Zymoclean Gel DNA Recovery Kit (Zymo Research).
After quantification by Qubit (Invitrogen), barcoded samples were mixed and sequenced on an Illumina HiSeq3000 system.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
RNA-seq human CD8+ T IL7+IL15 24h donor1
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| Data processing |
Sequenced reads were aligned to the mm10 genome assembly using Bowtie 2.2.6 and Tophat 2.1.1
Aligned reads are converted to BAM files using samtools v0.1.8. BAM files are subsequently converted to BED files using bedtools v2.25.0. MACS v1.4.2 is used to call peaks using the BED files. Unpublished python scripts were used to calculate RPKM (reads per kilobase per million reads) to normalize gene expression values for RNA-Seq data. R package edgeR was used to identify differentially expressed genes or differential ATAC-Seq peaks among different conditions.
Genome_build: mm10 and hg19
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| Submission date |
Dec 15, 2020 |
| Last update date |
Jul 07, 2021 |
| Contact name |
Peng Li |
| E-mail(s) |
peng.li@nih.gov
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| Organization name |
NIH
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| Department |
NHLBI
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| Lab |
LMI
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE138698 |
An engineered IL-2 partial agonist promotes CD8+ T cell stemness |
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| Relations |
| BioSample |
SAMN17085040 |
| SRA |
SRX9725121 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4976195_WJL2020_311.hg19.Donor1.CD8.IL15.24h.rpkm.gz |
915.0 Kb |
(ftp)(http) |
RPKM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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