Transcriptional profiling of activated CD4+ memory T cells exposed to anti-TNF and recombinant human TNF-alpha in vitro

We performed an RNA-seq analysis on FACS-sorted CD4+ memory T cells (Tmem, CD4+CD25-CD45RA-) stimulated in vitro with anti-CD3/CD28 coated beads (1:5), IL-2 (100 U/mL) in the presence of a TNF-alpha inhibitor (Etanercept, ETN, 5 µg/mL) or recombinant human TNF-alpha (rhTNF-alpha, 50 ng/mL). The cells were cultured for three days at 37°C and 5% CO2. On day three Tmem were lysed (Buffer RLT supplemented with DTT, QIAGEN) and RNA extraction was performed (RNeasy Plus Micro kit), followed by sample preparation with TruSeq (polyA) mRNA kits (Illumina), RNA sequencing (carried out on an Illumina HiSeq2500), and the alignment of trimmed fastQ files to GRCh38 human reference genome. We also performed a Quality control (QC). This was done using the tool FastQC FastQC/0.11.3-Java-1.7.0_80. QC metrics were calculated for the aligned reads using Picard-tools picard/1.130-Java-1.7.0_80, CollectRnaSeqMetrics, MarkDuplicates, CollectInsertSize-Metrics and SAMtools/1.2-foss-2015b flagstat. Finally, we carried out a Differential expression gene (DEG) analysis using DESeq2, in order to evaluate the impact of ETN and rhTNF-alpha on Transcriptome profiling of stimulated CD4+ Tmem. Principal Component Analysis (PCA) was carried out to visualize the samples given their entire transcriptome and any remaining batch effects.