Computational protocol: New insights into the heterogeneity of Th17 subsets contributing to HIV-1 persistence during antiretroviral therapy

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Protocol publication

[…] Matched memory CD4+ T-cell subsets were isolated by FACS from different HIV-uninfected donors stimulated with immobilized CD3 and soluble CD28 (1 µg/ml) for 4 days. Total RNA was isolated using RNeasy columns kit (Qiagen) according to the manufacturer’s protocol. RNA quantity was determined by Pearl nanophotometer (Implen, Germany) (106 cells yielded 1–5 µg RNA). Genome-wide analysis of gene expression was performed on total RNA by Génome Québec Innovation Centre (Montreal, Qc, Canada). Briefly, the quality of total RNA was tested using the Agilent 2100 Bioanalyzer chip. High quality RNA was reverse transcribed and hybridized on the Illumina HumanHT-12 v4 Expression BeadChip providing coverage for more then 47,000 transcripts and known splice variants across the human transcriptome. The expression of differentially expressed genes was identified as previously described []. The entire microarray dataset and technical information requested by Minimum Information About a Microarray Experiment (MIAME) are available at the Gene Expression Omnibus (GEO) database under accession number GSE66972. Differentially expressed genes (cut-off 1.3-fold; p < 0.05) were classified through Gene Ontology using the NetAffx web-based application (Affymetrix), while differentially expressed pathways were identified using Ingenuity Pathway Analysis (IPA) and Gene Set Variation Analysis (GSVA). Corresponding heat maps for biological function categories were generated using programming language R []. [...] Intracellular expression of cytokines was measured by FACS [] using the BD Cytofix/Cytoperm kit (BD Biosciences) and specific Abs (See table above). Analysis was performed using Diva and FlowJo. Polyfunctional profiles were analyzed using the SPICE (version 5.3; provided by Mario Roederer, VRC/NIAID/NIH) []. […]

Pipeline specifications

Software tools IPA, FlowJo, SPICE
Application Flow cytometry
Organisms Human immunodeficiency virus 1, Human immunodeficiency virus 2, Candida albicans