Geneatlas system

Total RNA was extracted from whole blood samples and treated to deplete for globin mRNA according to the protocol described by Gille et al. [33 (link)]. Whole genome transcript profiling was then performed with HG-U219 oligonucleotide expression probe arrays (Affymetrix, USA). Preparation of samples for profiling including reverse transcription, amplification, amplified RNA labelling, purification, fragmentation, and hybridization steps were performed as defined previously [33 (link)]. Arrays were measured in accordance with manufacturer’s recommendations using Affymetrix GeneAtlas™System. Raw array data was imported to the R environment (version 4.1.2) [34 ] and corrected for background noise, log2 transformed, normalized for inter-array variation (quantile normalization) [35 (link)] and summarized using the rma (Robust Multichip Average) function from affy (version 1.72.0) [36 (link)]. In addition, probe sets were filtered to keep only the most variable probe set per gene (based on standard deviation), probe sets assigned to gene symbols (hgu219.db, version 3.2.3) [37 ] and probe sets with average expression (log2) > 5. Of the 49′386 probe sets, 6′128 probe sets were retained for further analysis.

RNA was extracted from 1 ml of heparinized peripheral whole blood collected from the participants before inoculation and at seven time points after inoculation (3, 24 and 48 hours, and one, two, four and seven weeks). After purification with the QIAamp RNA Blood Mini Kit (Qiagen), 100 ng of RNA was amplified using GeneChip 3´IVT Express Kit, after fragmentation and labeling, aRNA was hybridized onto Human Genome U219 arrays (all Affymetrix) for 16 hours at 45°C, either by Aros Applied Biotechnology or in-house using the GeneAtlas system (Affymetrix). Transcriptomic data was normalized using Robust Multi Average (RMA) implemented in the Partek Express software. Fold change was calculated by comparing each sample to the pre-inoculation samples in each individual. Genes with absolute fold change >2.0 were considered differentially expressed. Heat-maps were constructed using the Gitools software. Differentially expressed genes and regulated pathways were analyzed using the Gene Set Enrichment Analysis (GSEA, Broad Institute) and the Ingenuity Pathway Analysis (IPA, Qiagen Bioinformatics) softwares. Fimbriae-specific effects on transcription were distinguished by comparing the response to E. coli 83972 inoculation at each time point and in each patient.

Microarray analysis was performed as described previously [13 (link)]. Briefly, a GeneAtlas® system (Affymetrix, Santa Clara, CA, USA) was employed. RNA extraction and quality control were performed as described previously [13 (link)]. Labeled fragmented single-stranded cDNA (ss-cDNA) was synthesized by using purified total RNA (100-500 ng) as the template following Affymetrix WT PLUS Labeling Assay protocol. Then, the mixtures of biotinylate labeled ss-cDNAs were hybridized onto porcine Gene 1.1 ST Arrays (Affymetrix, Santa Clara, CA, USA). After being washed by a fluidic station, the arrays were scanned with an imaging station in a GeneAtlas® system (Affymetrix, Santa Clara, CA, USA), and the acquired array raw data were analyzed with the Affymetrix Command Console Software Version 1.4. Quantile normalization and subsequent data processing were performed by the Affymetrix Transcriptome Analysis Console (TAC) Software 4.0. The cutoffs for differentially expressed genes (DEGs) were set at fold change > 1.5 or <0.67 and FDR < 0.01. Gene Ontology (GO) (http://www.geneontology.org/) and Reactome pathway database (http://www.reactome.org) were used for further analysis.

The microarray study was carried out according to previously described protocols [27 (link),28 (link),29 (link)]. The isolated RNA was pooled into fifteen samples, representing wild type littermates (n = 3) and R6/1 mice (n = 4), at the symptomatic stage of 6 months of age; wild type littermates (n = 4) and R6/2 mice (n = 4), at the symptomatic stage of 9 weeks of age. The total RNA (100 ng) from each sample was subjected to two rounds of sense cDNA amplification, biotin labelling, and fragmentation according to the manufacturer’s protocol. (GeneChip^® WT Plus Reagent Kit, Affymetrix, Santa Clara, CA, USA). Biotin-labelled fragments of cDNA (5.5 μg) were hybridised to the Affymetrix^® Mouse Gene 2.1 ST Array Strip (45 C/20 h). After hybridisation, array strips were washed and stained by the Fluidics Station of a Gene Atlas System (Affymetrix). Next, the array strips were scanned by the Imaging Station from a Gene Atlas System. Preliminary analysis of the scanned microarrays was performed using Affymetrix Gene Atlas TM Operating Software (Affymetrix, Santa Clara, CA, USA). The quality of gene expression data was checked according to quality control criteria provided by the software. The obtained CEL files were imported into downstream data analysis.

A series of seven mice (three Cd19^Cre and four Myd88^L252P) was studied in parallel with bone marrow purified tumor B-cells from a series of 11 patients with MYD88^L265P WM (series 1) as well as lymph nodes from a series of 58 patients: 19 MYD88^WT chronic lymphocytic leukemia, 15 MYD88^L265P WM, 12 MYD88^wt Nodal marginal zone lymphoma, 5 MYD88^wt WM, 4 follicular lymphoma and 3 patients with benign follicular hyperplasia (series 2, Supplementary Tables 1 and 2). Approval of this protocol was obtained from the local IRB of the CHRU of Lille (CSTMT043). MYD88 and CXCR4 mutational status was determined as previously described (6 (link)). Total mRNA was extracted from whole infiltrated tissues and purified B-cells as reported (24 (link)). For humans and mice, RNA amplification and hybridization onto microarrays were performed on an Affymetrix Human Genome U133 Plus 2.0 Array and on an Affymetrix Gene Atlas system^® with the MoGene-2_1-st-v1 Affymetrix chip (Affymetrix, Santa Clara, CA) respectively according to a previously described protocol (25 (link)) (GEO accession number GSE138273). Bioinformatic analyses are detailed in Supplementary Materials and Methods.

miRNA arrays were made using an miRNA 4.1 Array Strip Human (Affymetrix, Santa Clara, CA, USA). Affymetrix GeneAtlas system was used to scan the miRNA arrays. ll bioinformatics analyzes were performed using BioConductor. A detailed description of the miRNA microarray analysis and miRNA–RNA correlation was described in our previous articles [14 (link),26 (link)].

Microarray gene expression profiling with Mouse Genome 430 PM Strip arrays was performed following manufacturer recommendations (Affymetrix Inc, Santa Clara, CA) with specific adaptations based on the picoprofiling method [18 (link)]. Image intensities were extracted with Affymetrix GeneAtlas System software, normalized and summarized by RMA and analyzed for differential gene expression by Limma [19 ] with false discovery rate multiple testing significance correction. NK-related γδTCR, iNKT CD4⁺ and iNKT CD4^- cells specific gene sets [20 (link)] were tested by Gene Set Enrichment Analysis (GSEA) [21 (link)] for significant enrichment in CD11c⁺ T cells.