X ten pe150 platform

MiRNA deep sequencing was performed from the serum of the most typical subjects, including six HT-NC patients, six HT-CI patients, and three control individuals in Nanjing Geneseeq Inc., Nanjing, China. Briefly, serum exosomal total RNA quality was assessed using Agilent Bioanalyzer 2100. Then, the sequencing libraries were generated using NEBNext Multiplex Small RNA Library Prep Set (New England Biolabs, United States) following manufacturer’s recommendations, and index codes were added to attribute sequences to each sample. Libraries were pooled and sequenced on an Illumina X-ten PE150 platform. A minimum of 300 M reads was generated per sample.

Human liver total RNA was extracted with PureLink/Trizol, according to the manufacturer's instruction. The amount, quality and composition of isolated RNA were analyzed by Nanodrop, Qubit 3.0 for total RNA and an Agilent 2100 Bioanalyzer for total RNA integrity. RNA purity was checked using the NanoDrop One/One C (Thermo Fisher Scientific, CA, USA). RNA concentration was measured by Qubit® RNA HS Assay Kit in Qubit®3.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using the Agilent RNA 6000 Pico Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA-sequencing was performed by SHBio Inc., Shanghai, China. Sequencing libraries were generated using KAPA Stranded RNA-Seq Kit (KAPA, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Libraries were pooled and sequenced on an Illumina X-ten PE150 platform. A minimum of 6G data were generated per sample.