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 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.

丁酸钠（Sodium butyrate）与丁苯羟酸（bufexamac）处理激活态CD4+ T细胞，可削弱其新发HIV-1感染能力。本研究采用RNA测序（RNA-seq）获取处理后细胞的转录组变化信息，以解析其对HIV-1感染的调控效应。研究人员使用Easy-Sep™ Rosette人CD4+ T细胞富集试剂盒（加拿大Stemcell Technologies公司）从外周血单个核细胞（Peripheral Blood Mononuclear Cells, PBMC）中分离CD4+ T细胞。按照标准实验流程，使用白细胞介素-2（Interleukin-2, IL-2）与植物血凝素（Phytohemagglutinin, PHA）对分离得到的CD4+ T细胞进行5~7天的激活处理。为最小化供体差异带来的实验偏差，将4名健康供体的激活CD4+ T细胞混合，并将混合后的细胞分别用1.95 mM丁酸钠与0.04 mM丁苯羟酸处理，培养48小时；以未处理的细胞混合池作为空白对照。随后收集细胞，用磷酸盐缓冲液（PBS）洗涤后，按照操作说明书，使用Direct-zol RNA MiniPrep试剂盒（Zymo Research公司）从Trizol裂解液中分离总RNA；后续再采用Agencourt RNAClean XP磁珠（贝克曼库尔特公司）按照说明书进行额外纯化。本研究共构建了未处理、丁酸钠处理以及丁苯羟酸处理的细胞样本各3份独立生物学重复。通过Nanodrop进行定量后，使用Agilent 2100生物分析仪（Agilent Technologies公司）对RNA质量进行质控。选取RNA完整性数值（RIN）>7的优质总RNA，按照操作说明书，采用Lexogen的Illumina平台适配Sense mRNA Seq文库制备试剂盒V2，取500 ng总RNA构建测序文库。完成文库定量后，在Illumina HiSeq 1500测序平台上进行单端100 nt测序。测序数据在慕尼黑基因中心LAFUGA托管的Galaxy服务器上进行预处理：先进行解复用（demultiplexing），随后按照Lexogen官方流程进行序列修剪；将修剪后的序列比对至人类参考基因组hg19，比对工具采用STAR（版本2.5.2b-0）。利用HTSeq-count（版本1.0.0）对比对得到的有效读段进行计数，并通过DESeq（版本1.0.19）进行差异基因表达分析，设定错误发现率（False Discovery Rate, FDR）阈值为<0.01。