|
| Status |
Public on Jun 25, 2018 |
| Title |
HV_2-1_ET |
| Sample type |
RNA |
| |
|
| Source name |
HV n°2, triplicate 1, condition ET
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
cell type: PBMC
subject_number: HV2
sample_conditions: ET
sample_replicate: 1
sample_batch: 2
|
| Treatment protocol |
Citrated blood from healthy volunteers was obtained from EFS (Etablissement Français du Sang) and used immediately. According to EFS standardized procedures for blood donation, informed consent was obtained from healthy volunteers and personal data for blood donors were anonymized at time of blood donation and before blood transfer to our research lab. Peripheral blood mononuclear cells (PBMCs) were isolated from citrated venous blood with Unisep tubes density gradient centrifugation (Eurobio) and washed with PBS. Cell were cultured in 24-well plates at 2×106 cell/mL in X-Vivo 20 Medium (Lonza). As shown in Figure 3A, to induce the LPS-primed state, PBMCs were cultured in the presence or absence (control group) of 2 ng/mL LPS mix from Escherichia coli O55:B5, O127:B8 and O11:B4 (Sigma-Aldrich) and incubated overnight at 37°C and 5% CO2 (15 hours). Following a washing step, in order to induce reverse tolerance, PBMCs were incubated for an additional 24 hours in the presence or absence of 100 ng/mL of human IFN-g1b (Miltenyi Biotec). Finally PBMCs were stimulated a second time by adding 100 ng/mL of LPS for another six hours. To achieve a classical ex vivo endotoxin tolerance model, the step with IFNg stimulation is not carried out. For each conditions, supernatants were recovered and stored at -80°C. Cells were harvested, lysed in RLT buffer supplemented with β mercaptoéthanol and stored at -80°C until further processing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from PBMCs using RNeasy Plus Mini kits (Qiagen). For each RNA extraction, the residual genomic DNA was digested using the gDNA Eliminator spin column (Qiagen) and directly on RNeasy spin column using RNase-Free DNase Set (Qiagen). RNA was eluted in 30 µL of RNase Free Water. RNA quantity was determined for each sample using Nanodrop (Thermo Scientific) according to the manufacturer’s instructions. RNA quality was assessed on a Bioanalyser 2100 (Agilent) according to the manufacturer’s instructions. All samples have a RIN greater than 7.
|
| Label |
biotin
|
| Label protocol |
The cDNA synthesis and amplification steps were performed from 16 ng of RNA using the Ovation Pico WTA System V2 kit (Nugen). Briefly, cDNA synthesis was done using a mixture of random and polydT primers, followed by the synthesis of the complementary strand. The Single Primer Isothermal Amplification (SPIA) was then performed with hybrid DNA/RNA primers sensitive to RNAse-H digestion, in the presence of a DNA polymerase with strong strand displacement activity. The resulting amplified cDNA was purified using the QIAquick purification kit (Qiagen), from which, total DNA concentration was measured using the NanoDrop 1000 spectrophotometer (Thermo Scientific) and the product quality was checked on the Bioanalyser 2100 (Agilent).
|
| |
|
| Hybridization protocol |
5 micrograms of purified DNA were fragmented into 50-200 bp fragments and were 3-labeled using Encore Biotin Module kit (Nugen). The resulting target was mixed with standard hybridization controls and B2 oligonucleotides following recommendations of manufacturer. The hybridization cocktail was heat-denatured at 99°C for 2 minutes, incubated at 50°C for 5 minutes and centrifuged at 16,000 g for 5 minutes to pellet the residual salts. The HERV-V3 microarrays were prehybridized with 200 µL of hybridization buffer and placed under stirring (60 rpm) in an oven at 50°C for 10 minutes. The hybridization buffer was then replaced by the denatured hybridization cocktail. Hybridization was performed at 50°C for 18 hours in the oven under constant stirring (60 rpm). Washing and staining were carried out according to the protocol supplied by the manufacturer, using a fluidic station: GeneChip fluidic station 450 (Affymetrix).
|
| Scan protocol |
The arrays were finally scanned using a fluorometric scanner: GeneChip scanner 3000 7G (Affymetrix)
|
| Description |
HV n°2, triplicate 1, condition ET
|
| Data processing |
CEL files were transformed into matrix, normalized, adjusted for background noise (RMA normalization) and probes were summarized into probesets with command apt-probeset-summarize (V 1.18.0) with rma option . Batch effects were removed using COMBAT. Probesets were filtered when log2 signal was under 5.5 in more than 68% of samples.
|
| |
|
| Submission date |
Dec 18, 2017 |
| Last update date |
Jun 25, 2018 |
| Contact name |
Julien Textoris |
| E-mail(s) |
[email protected]
|
| Phone |
+33 472 119 546
|
| Organization name |
bioMérieux
|
| Department |
Medical Diagnostic Discovery Department (MD3)
|
| Lab |
Joint Research Unit - bioMérieux / HCL
|
| Street address |
Hôpital Edouard herriot - Pavillon P; 5 place d'Arsonval
|
| City |
Lyon |
| ZIP/Postal code |
69437 |
| Country |
France |
| |
|
| Platform ID |
GPL22462 |
| Series (1) |
| GSE108239 |
Analysis of HERV transcriptome in PBMCs upon dose dependent LPS stimulations identifies HERVs and genes shared signaling pathways |
|
