Unveiling immune interference: How dendritic cell response to co-infection with Aspergillus fumigatus is modulated by human cytomegalovirus and its virokine CMVIL10
Unveiling immune interference: How dendritic cell response to co-infection with Aspergillus fumigatus is modulated by human cytomegalovirus and its virokine CMVIL10
Monocyte-derived dendritic cells (moDCs) were generated from CD14+ monocytes isolated from peripheral blood mononuclear cell as previously described.  Seelbinder B, Wallstabe J, Marischen L, Weiss E, Wurster S, Page L, Löffler C, Bussemer L, Schmitt A-L, Wolf T, Linde J, Cicin-Sain L, Becker J, Kalinke U, Vogel J, Panagiotou G, Einsele H, Westermann AJ, Schäuble S, Loeffler J. 2020. Triple RNA-Seq Reveals Synergy in a Human Virus-Fungus Co-infection Model. Cell Reports 33:108389.  Guinan J, Lopez BS. 2020. Generating Bovine Monocyte-Derived Dendritic Cells for Experimental and Clinical Applications Using Commercially Available Serum-Free Medium. Frontiers in Immunology Volume 11 - 2020. Total RNA from the samples was isolated according to the manufacturer's instructions. An on-column DNase digestion step was included in the RNA isolation protocol. RNA extraction kit: RNeasy Mini/Micro Kit (Qiagen) Elution volume: 40 µl Library preparation was performed with the NEBNext® Poly(A) mRNA Magnetic Isolation Module and NEBNext® UltraTM II Directional mRNA kit (New England Biolabs) according to the manufacturer's instructions (2). The protocol starts with a poly(A) RNA selection step using Oligo dT Beads d(T). The mRNA was fragmented and samples were then introduced  into a reverse transcription to generate first strand cDNA. After the second strand cDNA  synthesis, 3'-ends were adenylated and sequencing adapters were ligated. Those comprise  sequencing primer- and flow cell-binding sites, as well as indices for multiplexed sequencing  of pooled libraries. Adapter-ligated fragments were amplified during a 15-cycle PCR reaction. Universal Human Reference RNA (UHR) was simultaneously processed as internal positive  control (PosC) but discarded after successful library validation.