RNA-seq raw count matrix

HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C. RNA was harvested using Rneasy mini plus kit (Qiagen). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 3 ug of total RNA was used for the construction of sequencing libraries. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. RNA-seq raw reads of fastq format were firstly processed through in-house perl scripts. Index of the reference genome (hg38) was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. featureCounts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene.

本研究中，HEK293细胞于添加10%胎牛血清（FBS）及抗生素的DMEM培养基中，在37℃、含5%二氧化碳的湿润培养环境内维持培养。采用RNeasy Mini Plus试剂盒（Qiagen）提取总RNA；使用美国加利福尼亚州安捷伦科技（Agilent Technologies）的2100生物分析仪专用RNA Nano 6000检测试剂盒评估RNA完整性。取3 μg总RNA用于测序文库构建，按照制造商的操作指南，使用适配Illumina®平台的NEBNext® UltraTM RNA文库制备试剂盒（NEB，美国）完成文库制备。首先通过自研Perl脚本处理fastq格式的RNA-seq原始读段；使用Hisat2 v2.0.5构建参考基因组（hg38）的索引，并通过该工具将双端清洁读段比对至参考基因组。采用featureCounts v1.5.0-p3统计比对至每个基因的读段数量，随后基于基因长度及比对至该基因的读段计数，计算每个基因的FPKM值。