The total RNA was extracted from the blood sample using the RNAliquid Reagent according to the manufacturer's protocols. Agilent 2100 was utilized to test the RNA integrity number (RIN) (7). RNA conditions to be met for sequencing were as follows: (1) the total amount (required for single database construction) is 5 μg; (2) the concentration was ≥200 ng/μl; and (3) the value of OD260/280 was between 1.8 and 2.2. After total RNA DNase I digestion, removal of rRNA, RNA interruption, synthesis of reverse transcriptional one and two strands, end repair, cDNA with an “A” at the 3′ end, connection of cDNA 5′ adapter, digestion of two strands of cDNA, PCR reaction, and product recovery and library quality inspection, the Illumina Hiseq x-ten platform (PE150 strategy) (8) was used to perform RNA sequencing for lncRNA. After RNA fragment selection, 3' connector connection, reverse primer annealing, 5' connector connection, the synthesis of a strand of cDNA, PCR amplification, library fragment selection, library quantification, and pooling annularization, BGISEQ-500 platform (SE50 strategy) (9) was used to perform RNA sequencing for mRNA. The Combat function for the SVA package was used to eliminate batch effect (10). The Fastx-Toolkit was used to trim 5' and 3' segments of reads to remove bases with mass <20 and delete reads with N >10%. For lncRNA analysis, the cleaned gene sequencing reads were aligned to the reference genome (GRCh38) for matching via HISAT2 (11) (https://ccb.jhu.edu/software/hisat2/index.shtml). Stringtie (12) (http://www.ccb.jhu.edu/software/stringtie/) was utilized to quantify the expression levels of lncRNA and mRNA. For mRNA analysis, the Rfam (13) was used for annotation analysis on measured small RNA. The mature mRNA and mRNA precursor sequences were downloaded from miRBase (14). The expression of mRNA was quantified with miRDeep2 (15). Finally, DEGSeq2 package (16) was used to compare the expression difference of lncRNA and mRNA between the two groups in the R environment. The value of fragments per kilobase of exon per million mapped reads (FPKM) of each gene/transcript in the sample was calculated according to the comparison results of all samples with the reference genome. The value was regarded as the expression amount of gene/transcript in the sample.

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