Gene Expression
Nebulas
Gene Expression NebulasSummary: We report the transcriptional profile of phosphatidylserine (PS) positive and PS negative non-naive peripheral blood CD8 T cells from patients infected with SARS-CoV-2. PS+ CD8 T cells showed increased expression of cell cyclce and cell division associated genes and reduced expression of genes regulating translational initiation, when compared to PS- CD 8 T cells. Furthermore, PS+ CD8 T cells show increased expression of effector T cell genes.
Overall Design: non-naive (CCR7+CD45RA-, CCR7-CD45RA-, CCR7-CD45RA+) PS+ and PS- peripheral blood CD8 T cells were sorted from 4 COVID-19 patients and analysed by RNAseq WHOmax: the maximal COVID-19 disease severity score of the patient, classified using the World Health Organization's (WHO) eight-point ordinal scale for COVID-19 trial endpoints.
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| Growth Protocol: | - |
| Treatment Protocol: | - |
| Extract Protocol: | Peripheral blood was collected into lithium heparin tubes (Sarstedt). PBMCs were isolated by Biocoll density gradient (Merck) centrifugation and then directly stained for FACS sorting. Live, Dump-, C8+,CCR7+CD45RA-, CCR7-CD45RA-, CCR7-CD45RA+, PS+ and PS- were directly sorted into TRIZOL on a FACSAriaFusion (BD) using a 100 μm nozzle. Total RNA was isolated and purified using Monarch columns (NEB). Poly(A)+ RNA was isolated from the total RNA sample. First-strand cDNA synthesis was primed with a N6 randomized primer. After fragmentation, the Illumina TruSeq sequencing adapters were ligated in a strand specific manner to the 5' and 3' ends of the cDNA fragments. In this way, a strand specific PCR amplification of the cDNA was achieved using a proof-reading enzyme. For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool in the size range of 250-700 bp was eluted from a preparative agarose gel. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA size range was 250-700 bp. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 1x75 bp read length. |
| Library Construction Protocol: | First-strand cDNA synthesis was primed with a N6 randomized primer. After fragmentation, the Illumina TruSeq sequencing adapters were ligated in a strand specific manner to the 5' and 3' ends of the cDNA fragments. In this way, a strand specific PCR amplification of the cDNA was achieved using a proof-reading enzyme. For Illumina NextSeq sequencing, the samples were pooled in approximately equimolar amounts. The cDNA pool in the size range of 250-700 bp was eluted from a preparative agarose gel. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA size range was 250-700 bp. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 1x75 bp read length. |
| Molecule Type: | polyA(+) RNA |
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| Library Layout: | SINGLE |
| Library Strand: | Reverse |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina NextSeq 500 |
| Strand-Specific: | Specific |