Hiseq 2500 sequencing analysis

Blood samples were drawn from HCC patients. RNA extraction was performed by miRNeasy Kit (Qiagen, Hilden, Germany), according to the manufacturer’s protocol. The resultant product was quantified using the Nanoquant Infinite M200 Microplate Reader (Tecan, Männedorf, Switzerland).
Total miRNA was isolated through polyacrylamide gel electrophoresis purification after a pair of adaptors was ligated to their 3’ and 5’ ends. Amplification of the miRNA (21 cycles) was performed, followed by isolation and gel purification of the fragments around 90 bp to prepare the yield for Illumina HiSeq 2500 sequencing analysis, according to the manufacturer’s instructions. Finally, data were computationally analyzed.

Microbial DNA was extracted using HiPure Soil DNA Extraction Kit (Magen, Guangzhou, China) following the manufacturer’s protocol. DNA extracts as templates were stored at −20 °C for later PCR amplification. PCR mixture preparation, reaction conditions, and further product purification were conducted following the procedures described in Huang et al. (2022) . The purified PCR amplicons were sent to Gene Denovo Biotechnology Co. Ltd. (Guangzhou, China) for Illumina HiSeq 2500 sequencing analysis. The sequencing raw data was filtered with operational taxonomic unit (OTU) annotation operations, consistent with our previous study (Huang et al., 2022 ).