Next-generation sequencing facilitates quantitative analysis of transcriptome profile in IEC 4.1 cells in response to CSpV1 RNA transfection

The Cryptosporidium parvum virus 1 (CSpV1) is a member of the family Partitiviridae, genus Cryspovirus that infects C. parvum. This study aims to measure the impact of CSpV1 on the transcriptome profile in cultured IEC 4.1. Cells were treated with specific dsRNAs encoding CSpV1 viral capsid protein (CA) and RNA dependent RNA polymerase (RdRp). Cells treated with a scrambled non-specific siRNA were used as the control. Total RNA was collected for the genome-wide analysis, for sequencing using BGISEQ-500 platform. The IEC 4.1 murine intestinal epithelial cells were grown to 80% confluence for four groups: the siRNA control (Group B, cells treated with a non-specific scrambled siRNA control), the dsCA transfected (Group C, cells treated with the double-strand RNA encoding CSpV1 capsid protein), dsRdRp transfected (Group D, cells treated with the double-strand RNA encoding CSpV1 RNA dependent RNA polymerase), and dsCA/dsRdRp transfected (Group F, cells treated with double-strand RNA encoding CSpV1 capsid protein plus double strand RNA encoding RNA dependent RNA polymerase). Cells were treated with the transfection for 24h. Total RNAs were prepared with the RNeasy Mini kit (Qiagen) according to the manufacturer’s instruction.

微小隐孢子虫病毒1型（Cryptosporidium parvum virus 1，CSpV1）隶属于双分病毒科（Partitiviridae）隐孢子虫病毒属（Cryspovirus），可感染微小隐孢子虫（C. parvum）。本研究旨在探究CSpV1对体外培养的IEC 4.1细胞转录组谱的影响。实验中，我们使用编码CSpV1病毒衣壳蛋白（CA）与RNA依赖RNA聚合酶（RNA dependent RNA polymerase，RdRp）的特异性双链RNA（double-stranded RNA，dsRNA）处理细胞，并以乱序非特异性小干扰RNA（small interfering RNA，siRNA）处理的细胞作为对照。收集总RNA进行全基因组分析，采用BGISEQ-500平台完成测序。实验所用的IEC 4.1小鼠肠上皮细胞培养至80%汇合度，分为四组：siRNA对照组（B组，经非特异性乱序siRNA处理的细胞）、dsCA转染组（C组，转染编码CSpV1衣壳蛋白的双链RNA的细胞）、dsRdRp转染组（D组，转染编码CSpV1 RNA依赖RNA聚合酶的双链RNA的细胞）以及dsCA/dsRdRp共转染组（F组，同时转染编码CSpV1衣壳蛋白与RNA依赖RNA聚合酶的双链RNA的细胞）。转染处理24小时后，按照制造商提供的操作说明书，使用RNeasy Mini试剂盒（Qiagen）提取总RNA。