Transcriptional profiling of wild type Neurospora crassa cells treated with novel antifungal agents. Neurospora crassa

Purpose: Evaluate the transcriptional profile of Neurospora crassa cells treated with a group of novel antifungal compounds Methods: Conidial suspensions were obtained and 1 x 106 cells/ml incubated in minimal medium for 6 hours (26ºC, 140 rpm, constant light) followed by the addition of the indicated compound (or DMSO) and growth for 1 more hour. Cells were harvested using 0.45 μm filters and immediately frozen in liquid nitrogen. Total RNA was isolated by the Trizol-Phenol-Chloroform method. After digestion of 25 μg RNA with TURBO DNAse (Life Technologies), mRNA was purified using Dynabeads oligo(dT) magnetic beads (Life Technologies). The mRNA was chemically fragmented using the Ambion RNA fragmentation kit (Life Technologies). First and second strand cDNA synthesis was achieved using appropriate kits (Life Technologies). The illumina TruSeq kit was employed to generate the cDNA libraries with indexing adapters essentially following the manufacturer’s protocol. After purification of the libraries with AMPure XP beads (Roche), the quality of the libraries was analysed in a Agilent 2100 Bioanalyzer. The cDNA libraries were sequenced in a illumina HiSeq2000 and single reads of 50 bp were obtained. Sequencing data was handled essentially with Tophat, Cufflinks and Cuffdiff. Expression levels are presented as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Results: XP13 promoted a particularly active transcriptional response. Functional enrichment analysis of the genes with altered expression revealed novel pathways that are affected by these compounds. Conclusions: These compounds are active in Neurospora crassa and we anticipate that this and future studies will ascertain their potential as antifungal agents. Overall design: The transcriptional profile of Neurospora crassa wild type cells exposed with a group of novel compounds with antifungal activity was analysed using illumina HiSeq2000 and single reads of 50 bp.研究目的：评估经一组新型抗真菌化合物处理的粗糙脉孢菌（Neurospora crassa）细胞的转录组谱。 实验方法：制备分生孢子悬液，将浓度调至1×10⁶个/mL，于基础培养基中在26℃、140转/分钟、持续光照条件下培养6小时；随后加入指定化合物（或二甲基亚砜（DMSO）），继续培养1小时。采用0.45μm滤膜收集细胞，立即置于液氮中速冻。总RNA通过Trizol-酚-氯仿法提取。取25μg RNA经TURBO DNA酶（Life Technologies公司）消化后，使用Dynabeads oligo(dT)磁珠（Life Technologies公司）纯化mRNA。采用Ambion RNA片段化试剂盒（Life Technologies公司）对mRNA进行化学片段化。使用配套试剂盒（Life Technologies公司）完成第一链与第二链cDNA合成。采用Illumina TruSeq试剂盒，严格遵循制造商操作流程构建带有索引接头的cDNA文库。使用AMPure XP磁珠（Roche公司）纯化文库后，通过Agilent 2100生物分析仪检测文库质量。将cDNA文库在Illumina HiSeq2000测序平台上进行测序，获得50bp单端读段（reads）。测序数据主要通过Tophat、Cufflinks及Cuffdiff进行分析处理。基因表达水平以每百万比对reads的转录本每千碱基片段数（Fragments Per Kilobase of transcript per Million mapped reads，FPKM）表示。 实验结果：XP13可引发尤为强烈的转录应答。对表达差异基因进行功能富集分析后，发现了受此类化合物影响的全新信号通路。 研究结论：上述化合物在粗糙脉孢菌中具有生物活性，我们预计本研究及后续相关研究将明确其作为抗真菌药剂的应用潜力。 整体实验设计：本研究采用Illumina HiSeq2000测序平台及50bp单端读段测序技术，分析了经一组具有抗真菌活性的新型化合物处理的粗糙脉孢菌野生型细胞的转录组谱。