title: D11_0.02MOI_polyl25_ssON0.5
organism: Homo sapiens
description: Protocols: MoDC culture Monocytes were isolated from buffy coats using Ficoll centrifugation (Lymphoprep; Axis Shield) after negative selection using the RosetteSep Monocyte Enrichment Kit (StemCell Technologies). Monocyte-derived DC (MoDC) were obtained after six days of differentiation in complete RPMI medium (RPMI 1640, 1mM sodium pyruvate, 10mM HEPES, 2mM L-glutamine, 1% Penicillin/Streptomycin, Hyclone GE Healthcare, and 10% FBS, Sigma) complemented with GM-CSF (250ng/mL; PeproTech) and rIL-4 (6.5ng/mL; R&amp;D Systems). Cells were seeded at a density of 5x10 5 cells/mL and after three days of differentiation, 50% of the medium was replaced and new cytokines added. Staining for flow cytometry was done before and at indicated time points post viral infection. IAV infection and treatments Stock of pandemic H1N1 virus strain A/Cal07/2009 was kindly provided by Bertin-Pharma, France. MoDC were mock-exposed or exposed to IAV or heat inactivated IAV (HI IAV,30min at 56°C) at a multiplicity of infection (MOI) of 0.02, 0.2 or 2 for 4h at 37°C 5%CO 2 in serum-free RPMI medium, washed in pre-warmed complete RPMI medium and distributed in 24 wells plates (0.5x10 6 /mL). Cells were then treated or not with the following molecules: synthetic, endotoxin-free, completely phosphorothioate-modified oligonucleotides named ssON (0.5µM; Integrated DNA Technologies), high molecular weight PolyI:C (25µg/mL; Invivogen) or the combination of both, referred as ssON/PolyI:C. The sequence of ssON is: 5'-GAAGTTTTGAGGTTTTGAAGTTGTTGGTGGTGGTG-3'. At 24h or 48h post infection (p.i), supernatants and MoDC were harvested and kept at -80°C until RNA purification was performed. RNeasy ® Plus Mini kit (QIAGEN) was used for total cells RNA extraction while QIAamp ® Viral RNA mini kit (QIAGEN) was used for viral RNA purification in supernatants, following manufacturer' spin protocol. Illumina TruSeq Stranded total RNA (ribosomal depletion)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-05-03T04:03:17Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-04-23T11:05:57Z
attributes.tag: External Id
attributes.value: SAMEA5574168
attributes.tag: INSDC center name
attributes.value: Royal Institute of Technology - KTH, Sweden
attributes.tag: INSDC first public
attributes.value: 2019-05-03T04:03:17Z
attributes.tag: INSDC last update
attributes.value: 2019-04-23T11:05:57Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7803:D11_0.02MOI_polyl25_ssON0.5
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: dendritic cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: individual
attributes.value: D11
attributes.tag: organism part
attributes.value: blood
attributes.tag: sample name
attributes.value: E-MTAB-7803:D11_0.02MOI_polyl25_ssON0.5
attributes.tag: scientific_name
attributes.value: Homo sapiens