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SRX9718712: GSM4984122: BR.a4; Homo sapiens; RNA-Seq
1 ILLUMINA (NextSeq 500) run: 1.1M spots, 69.4M bases, 27Mb downloads

Submitted by: NCBI (GEO)
Study: Human plasmacytoid dendritic cells mount a distinct antiviral response to virus-infected cells
show Abstracthide Abstract
Plasmacytoid dendritic cells (pDCs) can rapidly produce interferons and other soluble factors in response to extracellular viruses or virus mimics such as CpG-containing DNA. pDCs can also recognize live cells infected with certain RNA viruses, but the relevance and functional consequences of such recognition remain unclear. We studied the response of primary DCs to the prototypical persistent DNA virus, the human cytomegalovirus (CMV). Human pDCs responded poorly to free CMV but strongly to live CMV-infected fibroblasts, in a process that involved integrin-mediated adhesion, transfer of viral DNA to pDCs and its recognition through TLR9. Compared to transient polyfunctional responses to CpG or free influenza virus, pDC response to CMV-infected cells was long-lasting, dominated by the production of type I (IFN-I) and type III (IFN-III) interferons, and lacked diversification into functionally distinct populations. Similarly, pDC activation by influenza-infected lung epithelial cells was highly efficient, prolonged and dominated by interferon production. Prolonged pDC activation by CMV-infected cells facilitated the activation of natural killer cells that are critical for CMV control. Finally, patients with CMV viremia harbored phenotypically activated pDCs and increased levels of IFN-I and IFN-III in circulation. Thus, recognition of live infected cells is a common mechanism of virus detection by pDCs that elicits a unique antiviral response program. Overall design: RNA-sequencing of pDCs treated with CpG, CMV, and both infected and uninfected MRC5 cells using PLATE-seq 3'-tag sequencing
Sample: BR.a4
SAMN17138067 • SRS7912868 • All experiments • All runs
Organism: Homo sapiens
Library:
Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: pDCs were detached from plates by gentle pipetting and stained for viability with the Zombie Aqua Fixable Vaiability Kit in PBS for 20 mins at 4C. Cells were washed in 4C medium and FAC-sorted to remove MRC5 cells based on forward and side-scatter using an SH800Z sorter (SONY Biotechnology). Total RNA from sorted live pDCs was isolated using an RNeasy Mini Kit (QIAGEN). Pooled RNA-seq was performed using the low-input RNA-seq protocol reported in Snyder et al, Science Immunology, 2019. All of the steps prior below that come before the barcoded cDNA libraries are pooled were performed using a BioMek 4000 automated liquid handling robot (Beckman). Briefly, we added deoxyribonucleotides (dNTPs) (ThermoFisher) to a final concentration of 2 mM and reverse transcriptase primer to 2 uM followed by primer annealing at 72C for 3 mins. We then added 7.5 uL of reverse transcription mixture to each well comprised of 2M betaine (Affymetrix), 2x ProtoScript Buffer (NEB), 12 mM MgCl2 (ThermoFisher), 10 mM dithiothreitol (ThermoFisher), 5.3 U of Protoscript II reverse transcriptase (NEB), 0.53 U of SUPERaseIN (ThermoFisher), and 2 uM template-switching oligo. The reverse transcription reaction was incubated at 42C for 90 mins followed by 10 cycles of 50C for 2 mins, 42C for 2 mins, and 70C for 10 mins. Next, 2 uL of exonuclease I (ThermoFisher) was added to each well (1.875 U of exonuclease I in water) and the resulting mixture was incubated at 37C for 30 mins, 85C for 15 mins, and 75C for 30s.
Experiment attributes:
GEO Accession: GSM4984122
Links:
Runs: 1 run, 1.1M spots, 69.4M bases, 27Mb
Run# of Spots# of BasesSizePublished
SRR132896201,058,96469.4M27Mb2021-03-01

ID:
12709360

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