Homo sapiens
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)
ENA-FIRST-PUBLIC
2019-05-03T04:03:17Z
ENA-LAST-UPDATE
2019-04-23T11:05:57Z
External Id
SAMEA5574184
INSDC center name
Royal Institute of Technology - KTH, Sweden
INSDC first public
2019-05-03T04:03:17Z
INSDC last update
2019-04-23T11:05:57Z
INSDC status
public
Submitter Id
E-MTAB-7803:D11_0.2MOI_polyl25
broker name
ArrayExpress
cell type
dendritic cell
common name
human
developmental stage
adult
disease
normal
individual
D11
organism part
blood
sample name
E-MTAB-7803:D11_0.2MOI_polyl25
scientific_name
Homo sapiens