RNA Sequencing

Total RNA was extracted using QIAGEN RNeasy Kit (QIAGEN, Hilden, Germany). Library preparation was conducted at BGI following the guide of the standard protocol. Library preparation (BGISEQ-500RS High-throughput sequencing kit, PE50, V3.0, MGI Tech Co., Ltd., Shenzhen, China), hybridization, and sequencing were performed according to the manufacturer’s procedure from BGI (BGI-Shenzhen, China). The sequencing was performed at BGI-Shenzhen using the BGISEQ-500 system. The sequencing data were filtered to remove very low-quality reads using SOAP nuke (v1.5.2) software. The processed FASTQ files were mapped to the human transcriptome and genome using HISAT2 (v2.0.4). The genome version was GRCh38, with annotations from Bowtie2 (v2.2.5). The expression level of the gene was calculated by RSEM (v1.2.12). Differential expression was done with DEseq2 (v1.4.5) package. To take an insight into the change of phenotype, KEGG² enrichment analysis of annotated differentially expressed genes (DEGs) were performed by Phyper³ based on hypergeometric test. Significant DEGs were defined as ones with at least 0.3 fragments per kilobase million (FPKM) level of expression in at least one of the conditions and a q-value less than 0.05 by Bonferroni test.