Transcriptome analysis of activated CD4+ T cells treated either with sodium butyrate or bufexamac

Sodium butyrate and bufexamac treatment of activated CD4+ T cells impairs de novo HIV-1 infection. RNAseq was used to obtain information about the cellular changes upon treatment, which could explain the observed effects on HIV-1 infection Overall design: CD4 T cells were isolated from PBMCs using Easy-SepTM Rosette Human CD4+ T cell enrichment kit (Stemcell Technologies, Canada). Isolated cells were activated with IL-2 and PHA for 5-7 days according to standard protocols. To minimize donor effects, activated T cells from four donors were pooled and activated CD4+ T cell-pools were treated either with 1.95 mM sodium butyrate or 0.04 mM bufexamac and cultured for 48 h. Untreated T cell-pools served as control. Subsequently, cells were harvested, washed with PBS, and RNA was isolated from trizol lysates of cells using Direct-zol RNA Miniprep Kit (Zymo Research) following manufacturer's instructions. Isolated RNA was additionally purified using Agencourt RNAClean XP beads (Beckman Coulter) following manufacturer's instructions. In total 3 independent samples of untreated, sodium butyrate treated , and bufexamac treated cells were generated. After quantification (Nanodrop), the RNA was quality controlled using a Bioanalyzer (Agilent Technologies). Good quality RNA (RIN > 7) was used to generate sequencing libraries by using 500 ng total RNA in a Sense mRNA Seq Libarary Prep Kit V2 for Illumina platforms (Lexogen) following manufacture's instructions. Libraries were quantified and subsequently sequenced on an Illumina HiSeq 1500 (sequencing mode: 100 nt, single-end). The sequencing data was preprocessed on a Galaxy server (hosted by LAFUGA, Gene Center, Munich). After demultiplexing, the data was trimmed according to Lexogen. The output was mapped to the human genome (hg19) using STAR (v. 2.5.2b-0). Abundant reads were further analyzed using HTSeq-count (v. 1.0.0) and a differential gene expression analysis was performed using DESeq (v. 1.0.19) setting the FDR < 0.01.