Encore biotinil module

For primary cell experiments, cell pellets for mRNA isolation were lysed in Trizol (Ambion, Life Technologies) and RNA extracted by guanidium-isothiocyanate phenol/chloroform extraction method following the manufacturer’s instructions. Total RNA was quantified using the Nanodrop ND1000 and RNA integrity assessed with the Agilent Bioanalyzer 2100 using the RNA 6000 Nano Chips. One hundred nanograms of total RNA was used to prepare the targets, using the 3′ IVT Express kit (Affymetrix) in accordance with the manufacturer’s instructions. Hybridisation cocktails were hybridised onto the Human Genome U133 plus2 gene chip (Affymetrix). For Jurkat cell line experiments, three technical replicates of Jurkat T cell stably expressing CYT-1 or CYT-2 and untransduced cells as control were analysed. RNA was converted to cDNA using the Ovation® Pico WTA Systems V2 and labelled using the Encore® BiotinIL Module (NuGen) according to the manufacturers recommendations. Labelled cDNA was hybridised onto Illumina HT12v4 arrays (Illumina).

Purified mRNA for the respective macrophage populations was isolated using the PureLink^® RNA Mini Kit (Ambion) according to the manufacturer’s protocol. The purity of the isolated mRNA was assessed using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific) and the quality and integrity using an Agilent 2100 Bioanalyzer (Agilent Technologies). mRNA was converted to cDNA, then subsequently amplified using the Ovation^® PicoSL WTA system V2 (NuGen), biotinylated using the Encore^® BiotinIL Module (NuGen) and then hybridised to MouseWG-6 V2.0 Beadchip microarray (Illumina). Following hybridisation, the arrays were washed, blocked and stained with streptavidin-Cy3 using the whole-genome gene expression direct hybridisation assay (Illumina). Microarrays were run on an Illumina iScan system, raw fluorescence signals were collected using GenomeStudio (Illumina), and the data imported into Partek Genomics Suite for analysis. Background was subtracted from the raw data and fluorescence signals were normalised using the quantiles method^{75 (link)}.

Fifteen nanograms of isolated RNA with the RNA Integrity Number of at least 6.5 (n=4 for Mrap2^+/+; n=3 for Mrap2^tm1a/tm1a) was converted into cDNA using Ovation PICO SL System V2 (NuGEN), which was then fragmented and labelled using Encore BiotinIL Module (NuGEN). About 1500ng of each labelled product was then hybridized with MouseRef-8v2.0 Expression BeadChip Kit according to the manual and scanned using iScan (Illumina, San Diego, CA, USA). Raw image data were converted to bsc format using Illumina GenomeStudio 2011.1 software. Bonferroni correction with Family-Wise Error Rate (FWER) of 0.05 was applied to identify statistical significance of gene expression changes. Pathway analysis was performed using DAVID6.7 (http://david.abcc.ncifcrf.gov/tools.jsp) and STRING 10 (http://string-db.org/).

RNA from colonic tissue fragments (distal region) was extracted using TRIsure (Bioline) as described above. Contaminating DNA was removed with the RNase-Free DNase Set (Qiagen) according to the manufacturer’s protocol. cDNA was synthetized using Ovation PicoSL WTA System V2 according to the manufacture’s protocol (Nugen, USA) and labeled using Encore BiotinIL module according to the manufacture’s protocol (Nugen, USA). RNA and cDNA quantity and quality were assessed using the Agilent RNA 6000 Nano Kit or Agilent RNA 6000 Pico Kit (depending on the amount of RNA) according to the manufacture’s protocol (Agilent Technologies, USA). Labeled cDNA were hybridized on a MouseWG-6 v2.0 Expression BeadChip (Illumina, USA).

Total RNA and small RNA were isolated from cells using the modified protocol of Ambion RNAqueous-Micro kit. Total RNA was measured by Nanodrop and and by Bioanalyzer using the Agilent RNA 6000 pico kit. Total RNA at 10ng was amplified with the NuGEN Ovation PicoSL WTA system to generate amplified cDNA, which was then labeled with Biotin (NuGEN Encore BiotinIL Module). Biotin labeled cDNA at 750ng obtained from CD8+ T cells of aged and young mice immunized with the AdC68-NP or AdC68-gDNP vaccine was hybridized to Illumina MouseWG-6 v2 whole genome BeadChips. All arrays were processed in the Wistar Institute Genomics Facility.

For total RNA Quantitative real time PCR and microArrays, neonates and adults were injected or not with 5 x 10⁵ or 5 x 10⁶ CFU of Lm-actA^-/- for 24h.
Total RNA from 40,000–60,000 sorted neonatal or adult CD8α^- DCs or adult CD8α⁺ DCs (collected from 40 to 60 neonates and 5 to 7 adults) was extracted with phenol/chloroform and purified with the RNeasy microkit (Qiagen) according to manufacturer’s instructions. For quantification of transcripts, reverse transcription and quantitative real-time PCR were performed in a single step using the TaqMan RNA Amplification (Roche Diagnostics) on a Lightcycler 480 apparatus (Roche Diagnostics). For individual samples, mRNA levels were normalized to those of β-actin. Sequence of primers and probes are available on request. For microArrays, total RNA was amplified using the Ovation PicoSL WTA System V2 (NuGen), labeled with biotin using the Encore BiotinIL Module (NuGen), and applied on Illumina HT12 bead arrays at the GIGA-GenoTranscriptomics platform (Liège, Belgium). Microarray data (derived from Affymetrix GeneChip arrays HG-U133 plus 2.0) from CD8α^- neonatal and CD8⁺ adult DCs samples were obtained from the National Center for Biotechnology Information Gene Expression Omnibus.