donor id: ND351 tissue: whole blood cell population: IL4 DC culture condition: H1N1 Brisbane dosage: MOI 5-1 time point: 12h
Treatment protocol
Monocyte-derived dendritic cell stimulation: At the end of cultures, cells were collected with a 60 mL syringe, washed in 1x PBS and resuspended in complete RPMI at 1x106 cells/mL. To increase throughput, stimulations were conducted in 96 1mL deep-well plates (Greiner Bio-One) in a total volume of 500uL (5x105 cells). All stimuli used are summarized in Table S1. At the end of the stimulation period, cells were spun at 350g for 7 minutes, washed with 1x PBS, spun again and lysed in 600uL (1x106 cells) or 350uL (5x105) of RLT buffer (#79216, Qiagen). Cell lysates were stored at -80°C until extraction. Monocyte-derived dendritic cell stimulation with pathogens: IL-4 and IFN-alpha DC were stimulated in vitro for 1, 2, 6, 12 or 24h with either live influenza virus H1N1 Brisbane (Multiplicity of Infection 5:1), heat-killed Salmonella enterica (108 cfu/mL, ATCC# 14028) or heat-killed Staphylococcus aureus (108 cfu/mL, Invivogen, #cat tlr-hksa) at 37°C, 5% CO2. All pathogen stimulations were conducted in 96-deep well plates as described above. At the end of incubation time, cells were washed twice with PBS, lysed in the plate 350 uL RLT buffer (RNEasy Kit, Qiagen) and stored at -80°C.
Growth protocol
Monocyte-derived dendritic cell cultures: Monocytes were obtained from frozen fraction 5 from healthy donor apheresis. Cells were thawed for one minute in a 37°C water bath and resuspended in 1x phosphate buffered saline (PBS). Cells were spun at 350g for 7 minutes, washed with 1x PBS, counted, washed again, and resuspended in PBS / 2% Fetal Bovine Serum (FBS) / 1mM EDTA at 5x107 cells/mL. Monocytes were then enriched using the EasySep Human Monocyte Enrichment Without CD16 Depletion Kit (#19058, StemCell Technologies) according to the manufacturer’s protocol. Once enriched, cells were counted and resuspended in serum-free CellGenix DC medium (#2005, CellGenix, Germany) / 1% Penicillin/Streptomycin at 1x106 cells/mL. GM-CSF (Leukine®, Genzyme Corporation) was added for all DC subsets at 200 ng/mL. For IL-4 DC, recombinant IL-4 was added at 50 ng/mL and cells were fed a full dose of GM-CSF and IL-4 at day 2 and day 4 of a 6-day culture. For IFN-alpha DC, IFN-alpha was added at 500U/mL, and cells were fed a full dose of GM-CSF and IFN-alpha at day 1 of a 3-day culture. Cell suspensions were injected into 72mL sterile culture bags (#2PF-0072-AC, AFC, Gaithersburg, MD) with a 60mL syringe and bags were closed with injection ports. Feeding of the cells during the culture was done with a 1mL syringe through the injection ports. Cells were cultured in an incubator at 37°C, 5% CO2. Influenza Virus propagation: Madin Darby Canine Kidney (MDCK) cells were grown to of 80% confluent in 75 cm flask. The cells were infected with A/Brisbane/59/2007 IVR 148 (H1N1) strain at a ratio of 1:100 for 1 hour. The cells were washed twice and incubated for 48 to 72 hours with media (1 X Opti-MEM). Supernatant was harvested post-infection. The virus titer from the supernatant was assessed by plague assay. Experiments were performed under BSL-3 laboratory conditions. Salmonella enterica culture: Bacteria were thawed and plated on a nutrient agar (Difco) plate with a sterile 10uL inoculating loop, incubated overnight at 37°C and subsequently stored at 4°C. A single colony was picked up from the agar plate and inoculated in 6mL Difco nutrient broth (in 15mL Falcon tube), and cultured overnight at 37°C on a shaker. After overnight expansion, bacter were brought in log phase by culturing 500uL of bacterial preparation in 4.5mL fresh broth for 1.5 hour at 37°C on a shaker. Bacteria concentration was measured using a standard curve, bacteria were heat-killed for 30 minutes in 72°C water bath and stored at -80°C until use.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cell lysates using the RNeasy Mini-Kit (#74104, Qiagen) according to the manufacturer's instructions. Following extraction, an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA) was used to measure RNA Integrity Numbers (RIN) for each sample. All samples with RIN values greater than seven were retained for further processing. RNA concentration was measured using a Nanodrop 1000 (Nanodrop Technologies, Wilmington, DE).
Label
biotin
Label protocol
250 ng of RNA from all samples passing quality control were amplified and labeled using the Illumina TotalPrep-96 RNA amplification kit (Ambion, Austin, TX).
Hybridization protocol
750 ng of amplified labeled RNA were hybridized overnight to Illumina HT12 V3 beadchips (Illumina, San Diego, CA).
Scan protocol
Following hybridization, each chip was washed, blocked, stained, and scanned on an Illumina BeadStation 500 following the manufacturer’s protocols.
Description
6142085028_I
Data processing
Matrix normalized data was Global Average Normalized per Illumina GenomeStudio and all signal <10 has been set to 10. The data was generated using in-house code that uses GenomeStudio's Global Avg. Normalization algorithm. It calculates the avg signal for each sample and the avg signal for the dataset. Then the signals are multiplied by the factor: (avg dataset signal/avg sample signal). The net result is that all sample average and the dataset average are equal. Matrix non-normalized data is the raw data computed from the chip by GenomeStudio.
