16s rRNA sequencing of Wolbachia-infected and uninfected Drosophila male gut

Wolbachia are the most widely distributed intracellular bacteria, and their most common effect on host phenotype is cytoplasmic incompatibility (CI). A variety of models have been proposed to decipher the molecular mechanism of CI, in which the HM (host modification) model predicts that the effectors of Wolbachia play an important role in sperm modification. However, due to the complexity of spermatogenesis and cell-type heterogeneity in the testis, we still do not know whether Wolbachia have different effects on cells at different stages of spermatogenesis, nor whether these effects are linked with CI. Therefore, we used single-cell RNA sequencing to analyze gene expression profiles in the adult male Drosophila testes with or without Wolbachia infection. We found that Wolbachia significantly affected the proportion of different types of germ cells and affected multiple metabolic pathways in germ cells. Most importantly, Wolbachia had the greatest impact on germline stem cells (GSCs), r..., One-day-old Wolbachia-infected (named as Wmale samples) and -uninfected male flies (named as Tmale samples) were used for intestinal genome extraction and 16S rRNA gene amplicon sequencing (see Supplement Text for details). For Wolbachia-infected and uninfected sample collection, flies are anesthetized on ice, washed with 75% ethanol and then rewashed two times with sterile water. The whole gut was dissected in sterile PBS, 10 individuals were collected as a sample and ten biological replicates were obtained for each line. Genomic DNA was extracted using Qiagen DNeasy blood and tissue kit (Qiagen, Germany). The V4 hypervariable regions of 16S rRNA gene were amplified by specific primers (forward: 5-GTGCCAGCMGCCGCGGTAA-3, reverse: 5-GGACTACHVGGGTWTCTAAT-3). All purified DNA products were prepared as a paired-end (2 x 250 bp) DNA library and sequenced on the Illumina HiSeq 2500 platform (Novogene, Tianjin, china). On average, 100,000 sequences were obtained from each sample., The raw data is the FASTQ file, which can be opened by any text editing software.

沃尔巴克氏体属（Wolbachia）是分布最广泛的胞内细菌，其对宿主表型最常见的影响为细胞质不相容性（cytoplasmic incompatibility, CI）。目前已提出多种模型以解析CI的分子机制，其中宿主修饰（host modification, HM）模型预测沃尔巴克氏体的效应因子在精子修饰过程中发挥重要作用。然而，由于精子发生过程复杂且睾丸内细胞类型具有异质性，目前我们仍不清楚沃尔巴克氏体对精子发生不同阶段的细胞是否存在差异化影响，亦不清楚这些影响是否与CI相关。因此，本研究采用单细胞RNA测序（single-cell RNA sequencing）分析了感染与未感染沃尔巴克氏体的成年雄性果蝇睾丸中的基因表达谱。结果发现，沃尔巴克氏体可显著影响不同类型生殖细胞的比例，并对生殖细胞内的多条代谢通路产生调控作用。最为关键的是，沃尔巴克氏体对生殖系干细胞（germline stem cells, GSCs）的影响最为显著……
本研究选取感染沃尔巴克氏体的一日龄雄果蝇（命名为Wmale样本）与未感染的一日龄雄果蝇（命名为Tmale样本）进行肠道基因组提取及16S rRNA基因扩增子测序（详细方法参见补充文本）。样本收集流程如下：将果蝇置于冰上麻醉，先用75%乙醇洗涤，再用无菌水重洗两次；在无菌磷酸盐缓冲液（PBS）中解剖完整肠道，每10只果蝇的肠道作为一个样本，每个品系设置10个生物学重复。采用Qiagen DNeasy血液与组织试剂盒（Qiagen，德国）提取基因组DNA。通过特异性引物（上游引物：5'-GTGCCAGCMGCCGCGGTAA-3'，下游引物：5'-GGACTACHVGGGTWTCTAAT-3'）扩增16S rRNA基因的V4高变区。将所有纯化后的DNA产物制备为双端（2×250 bp）DNA文库，并在Illumina HiSeq 2500测序平台（诺禾致源Novogene，中国天津）上完成测序。每个样本平均获得100,000条测序序列。
原始数据为FASTQ格式文件，可通过任意文本编辑软件打开。