Using single-worm RNA sequencing to study C. elegans responses to pathogen infection. Using single-worm RNA sequencing to study C. elegans responses to pathogen infection

We have developed a single-worm RNA-seq method to effectively profile gene expression in individual C. elegans and applied this method to study C. elegans responses to pathogen infection. Overall design: Five replicates of two groups of samples (five worms with or without exposure to P. aeruginosa for 24 hours) were collected and subjected to mechanical worm lysis, RNA isolation, cDNA synthesis, library preparation, sequencing, and sequence data analysis. At each step, the results of the single-worm RNA-seq were compared to those of standard RNA-seq. The sequence data (FASTQ files) of standard RNA-seq were generated in the study of Sellegounder et al. (Science Advances 2019; 5(11):eaaw4717) and were retrieved from the NCBI’s SRA database through the GEO using the accession number GSE122544. These sequence data (from N2_OP50 and N2_PA14 samples) were re-analyzed in the current study in the same way as in the single-worm RNA-seq.

本研究开发了单线虫RNA测序（single-worm RNA-seq）方法，可实现个体秀丽隐杆线虫（Caenorhabditis elegans, C. elegans）基因表达谱的高效表征，并利用该方法探究秀丽隐杆线虫对病原体感染的应答反应。

实验设计概述：本研究共收集两组样本各5次生物学重复，每次重复包含5只经铜绿假单胞菌（Pseudomonas aeruginosa, P. aeruginosa）感染24小时或未感染的秀丽隐杆线虫；随后依次开展线虫机械裂解、RNA提取、cDNA合成、文库构建、测序及序列数据分析工作。

在每一实验环节，均将单线虫RNA测序的结果与标准RNA测序的结果进行对比分析。

标准RNA测序的序列数据（FASTQ文件）源自Sellegounder等人2019年发表于《Science Advances》的研究（2019; 5(11):eaaw4717），并通过基因表达综合数据库（Gene Expression Omnibus, GEO）从国家生物技术信息中心（National Center for Biotechnology Information, NCBI）的序列读取档案（Sequence Read Archive, SRA）中以登录号GSE122544获取。

本研究采用与单线虫RNA测序一致的分析流程，对源自N2_OP50与N2_PA14样本的上述序列数据进行了重新分析。