Naïve and memory CD8+ T cells were generated using the OT-I system as described previously (32 (link),33 (link)). RNA-Seq was done essentially as described (34 (link)). Briefly, total RNAs were prepared using RNA-Tri (Bio&SELL) and further purified using the RNeasy mini kit (Qiagen) in combination with a DNase I (Qiagen) treatment. RNA sequencing libraries were prepared by using the TruSeq mRNA Library Preparation kit (Illumina). 125-bp paired-end reads were generated by using a HiSeq 2500 sequencer (Illumina) with V4 sequencing chemistry. Triplicate samples from naïve and memory T cells were sequenced (around 40 × 106 reads per sample) and analyzed using a MISO-based pipeline (35 (link)).
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Isolation and RNA-Seq of Naïve and Memory CD8+ T Cells
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Corresponding Organization : Freie Universität Berlin
Other organizations : Max Planck Institute for Molecular Genetics, Otto-von-Guericke University Magdeburg, Helmholtz-Zentrum Berlin für Materialien und Energie
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Variable analysis
independent variables
- Naïve CD8+ T cells
- Memory CD8+ T cells
- Gene expression profiles (measured by RNA-Seq)
- RNA extraction and purification methods (RNA-Tri, RNeasy mini kit, DNase I treatment)
- Library preparation (TruSeq mRNA Library Preparation kit)
- Sequencing platform (HiSeq 2500 sequencer with V4 sequencing chemistry)
- Triplicate samples from naïve and memory T cells were sequenced to ensure reproducibility.
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