The PBMC of patients was isolated from whole blood with density gradient centrifugation using separation medium (Ficoll-Paque PLUS, GM) and washed in PBS (Beijing Solarbio Science & Technology Co., China). Total RNA was extracted using TRIzol (Invitrogen Life Technologies, USA) and RNeasy Mini Kit (Qiagen company, GM). After extraction, the RNA purity was determined by a NanoPhotometer spectrophotometer (IMPLEN, CA, USA), the concentration was determined by a Qubit 2.0 Fluorometer (Life Technologies, CA, USA), and the integrity was determined by an RNA Nano 6000 Assay Kit in the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) from the Biomark Technologies company (Qingdao, China).
The transcriptome sequencing on extracted total RNA was conducted in Hiseq 2500 platform (Illumina, San Diego, CA, USA) and generated with paired-end reads. The adaptor sequences and low-quality raw reads were removed as quality control and transformed into a clean read. After data processing, the clean reads were mapped to the reference genome using TopHat2 software, and mapped reads would be annotated and further analyzed as detectable genes (40 (link)).
The transcriptome sequencing on extracted total RNA was conducted in Hiseq 2500 platform (Illumina, San Diego, CA, USA) and generated with paired-end reads. The adaptor sequences and low-quality raw reads were removed as quality control and transformed into a clean read. After data processing, the clean reads were mapped to the reference genome using TopHat2 software, and mapped reads would be annotated and further analyzed as detectable genes (40 (link)).