Human primeview array

Cells remaining after flow cytometry staining were lysed and mRNA and DNA extracted using the AllPrep DNA/RNA Mini Kit (Qiagen). Approximately 500 ng was sent to the Centre for Applied Genomics for analysis using the Affymetrix Human Primeview Array. Raw data were normalized by Robust Multichip Average (RMA) using the R package “simpleaffy,” and plate batch effects were removed using the “ComBat” function in the R package “sva”; transcripts below the 95th percentile of fluorescence for all background probes in more than 25% of arrays were removed prior to differential expression analysis. Gene expression differences from the mock treatment (n = 4, for each group) were estimated using the “limma” package in R; significant genes were identified as those with an FDR‐adjusted p‐value (i.e., q‐value) less than 0.05. Pathway analysis for significantly different genes was performed using the Reactome Pathway Browser, version 3.7 (https://reactome.org/PathwayBrowser/).

Expression studies were performed using the Affymetrix platform according to manufacturer’s instructions. Briefly, 1 μg total RNA was extracted with Trizol from MOs, DCs and MACs, and hybridized to an Affymetrix Human Prime View Array (Affymetrix Inc., Santa Clara, CA, USA). Probe intensity normalization and downstream analysis were obtained using statistical analysis language R in combination with Bioconductor repository functions (http://bioconductor.org). Normalized data obtained with the “affy” package algorithm vsnrma [37 (link)] was followed by probe identity filtering, under strong statistical confidence thresholds (p-value < 0.01; adjusted p value (BH) < 0.05; FC < 2 & FC > 2 for downregulated and upregulated respectively). Finally, comparison of expression and DNA methylation data were performed by applying custom R scripting.

As previously reported [21 ], the IRB Functional Genomics Core Faculty gave access to their microarray services, which included quality control testing of total RNA using Nanodrop spectrophotometry, and the Agilent Bioanalyser. cDNA libraries were constructed and amplified from 25 ng of total RNA with 17 cycles of amplification and using WTA2 (Sigma-Aldrich). 8 μg of the cDNA was cut into fragments by the enzyme DNAseI and biotinylated by a terminal transferase, which was obtained from the GeneChip Mapping 250K Nsp Assay Kit (Affymetrix). Following the Affymetrix protocol, a hybridization mixture was prepared with each sample being hybridized to a PrimeView Human array (Affymetrix). As detailed in the Fluidics protocol FS450_002, the arrays were washed and stained in Fluidics station 450 the samples were then scanned using the GeneChip scanner 3000 (both Affymetrix), following the recommendations of the manufacturer. The GCOS software permitted the generation of CEL files using DAT files (Affymetrix). The RMA algorithm 29 was used to determine the normalized expression intensities from Affymetrix CEL files, and microarray pre-processing was carried out using the R package (R core team (2014). Results in Supplementary Table 1 as previously reported [21 ].

Microarray services were provided by the IRB Functional Genomics Core Facility, including quality control tests of total RNA by Agilent Bioanalyzer and Nanodrop spectrophotometry. Briefly, cDNA library preparation and amplification were performed from 25 ng total RNA using WTA2 (Sigma-Aldrich) with 17 cycles of amplification. 8 µg cDNA were subsequently fragmented by DNaseI and biotinylated by terminal transferase obtained from GeneChip Mapping 250 K Nsp Assay Kit (Affymetrix). Hybridization mixture was prepared according to Affymetrix protocol. Each sample was hybridized to a PrimeView Human array (Affymetrix). Arrays were washed and stained in a Fluidics Station 450 (Fluidics protocol FS450_002) and scanned in a GeneChip Scanner 3000 (both Affymetrix) according to manufacturer’s recommendations. CEL files were generated from DAT files using GCOS software (Affymetrix). Normalized expression intensities were computed from Affymetrix CEL files using the RMA algorithm^{29 (link)}. Microarray pre-processing was performed using R package (R Core Team (2014) (see Supplementary Statistical Analysis section).