The global effect of the garlic compound Allicin on the changes in the transcriptome in Bacillus subtilis and Staphylococcus aureus USA300 analysed by RNA-seq of coding RNA

To monitor the global changes in gene expression of Staphylococcus aureus USA300 TCH1516 under exposure to the garlic compound Allicin by RNA-seq, cultivation was performed in shaking flasks in Luria Bertani (LB) medium in triplicate at 37°C until cells have reached an optical density at 540?nm of 2.0. Cells were harvested by centrifugation, washed with Belitsky minimal medium (BMM) and adapted to BMM for one hour before exposure to 300 µM Allicin stress. S. aureus cells of 3 replicate experiments were harvested before and 30 min after exposure to 300 µM Allicin and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. After precipitation with 3 M sodium acetate and isopropanol, the total RNA was washed with cold ethanol and the pellet was solved in sterile water. Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. TruSeq Stranded mRNA Library Prep Kit from Illumina (San Diego, CA, USA) was used to prepare cDNA libraries. The resulting cDNAs were sequenced paired end on an Illumina MiSeq system (San Diego, CA, USA) using 75 bp read length.

本数据集旨在通过RNA测序（RNA-seq）监测暴露于大蒜素（Allicin）的金黄色葡萄球菌（Staphylococcus aureus）USA300 TCH1516的全球基因表达变化。实验采用三重重复设计，于37℃下在摇瓶的LB培养基（Luria Bertani medium）中培养菌株，直至菌体在540nm波长下的光密度达到2.0。随后通过离心收集菌体，用贝利茨基基础培养基（BMM）洗涤菌体，并在BMM中适应性培养1小时后，施加300μM大蒜素胁迫。分别在施加300μM大蒜素胁迫前及胁迫30分钟后，收集三次重复实验的菌体样本；使用含3mM乙二胺四乙酸（EDTA）、200mM氯化钠（NaCl）的裂解液，通过Precellys24组织破碎仪对菌体进行裂解。采用苯酚-氯仿-异戊醇法进行RNA提取：经3M乙酸钠和异丙醇沉淀后，用冷乙醇洗涤总RNA沉淀，再将其溶于无菌水中。初始RNA质量通过比利时根特的Trinean Xpose检测系统，以及德国伯布林根安捷伦科技公司的安捷伦2100生物分析仪（搭载安捷伦RNA Nano 6000试剂盒）进行检测。对存在DNA污染的样本，使用凯杰（Qiagen）公司的脱氧核糖核酸酶（DNase）处理，随后按上述方法重新纯化，并再次通过Trinean Xpose和安捷伦生物分析仪复检。最终获得的RNA样本需满足无DNA污染、RNA完整性数（RIN）>9、23S/16S核糖体RNA比值>1.5。使用美国加利福尼亚州圣地亚哥因美纳（Illumina）公司的Ribo-Zero细菌核糖体RNA去除试剂盒，从提取的总RNA中去除核糖体RNA。去除效果通过安捷伦2100生物分析仪（搭载安捷伦RNA Pico 6000试剂盒）检测，确保样本无可检测到的核糖体RNA。采用因美纳公司的TruSeq链特异性mRNA文库制备试剂盒构建cDNA文库，最终将所得cDNA在因美纳MiSeq测序系统上进行双端测序，读长为75bp。