C08
Total RNA was extracted from about 2mL of each plasma sample using a commercially available miRNeasy Micro Kit (Qiagen, Germantown, MD, USA) according to the manufacturer's instructions. RNA quality and concentration from each extract were assessed by using an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). Approximately 200 ng of total RNA from each sample was used for library construction. Small RNA library preparation was performed with the TruSeq small RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the instructions. Briefly, 3' and 5' specific adapters were ligated onto the RNA 3' and 5' ends by using T4 RNA ligase, respectively. Then, reverse transcription followed by eleven cycle PCR was applied to create cDNA constructs based on the adapters-ligated RNA as the templates. The amplified cDNA was separated by 6% polyacrylamide gel electrophoresis (PAGE), and the target (~150nt small cDNA constructs which contain both 5' and 3' adapters) was retrieved and recovered by using the gel extraction method, and was subsequently concentrated by ethanol precipitation. The quality control of the small RNA library was validated by Agilent High Sensitivity DNA Kit (Agilent, Santa Clara, CA, USA) using Agilent 2100 Bioanalyzer. Total concentration of the library was quantified through Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Wilmington, DE, USA) using a Quantifluor-ST fluorometer (Promega, Madison, WI, USA). Finally, the valid concentration of the library was determined via qPCR method by using a StepOnePlus Real-Time PCR Systems (Applied Biosystems, Waltham, MA, USA). Single-end sequencing with 50-bp strategy was performed at a depth of 20M for one sample/lane in the Illumina HiSeq2500 platform.