title: GSM7830875: NC_13; Homo sapiens; RNA-Seq
library_construction_protocol: 10 mL fasting blood was withdrawn via venipuncture and stood in non-anticoagulant serum tube (BD Biosciences, USA) for 20 min at room temperature, followed by centrifugation at 1600 g for 10 min. Supernatant was transferred into a new Eppendorf tube, and stored at -80 oC immediately. Serum miRNA was isolated using QIAamp ccfDNA/RNA Kit (QIAGEN, Germany), and the concentration was detected by NanoDropTM One/OneC (ThermoFisher Scientific, USA), and the 260/280 quality was determined using Agilent Bioanalyzer 2100 (Agilent, USA). Qiaseq miRNA Library Kit (QIAGEN) was used for miRNA library construction. Briefly, 10 ng miRNA was diluted into 5 μL, and mixed with 1 μL RA3 adaptor (10 mM) at 70 oC for 2 min, then rapidly cooled on ice. Mixture was incubated with 2 μL ligation buffer, 1 μL RNase Inhibitor, 1 μL T4 RNA Ligase at 28 oC for 1h, and 1 μL Stop Oligo for another 15 min. RA5 adaptor containing with three parts (S1, S2 and S3) was modified and synthesized (Sangon Biotech, China). S1 was a universal connector provided by kit. S2 consisted of 15 random bases, which could produce 1.07 billion complexity to accommodate every miRNA molecule one by one. The purpose of RA5 adaptor was to remove the reads of the identical S2 and miRNA in the subsequent data analysis, because they came from the repeats generated by PCR in the process of library construction and sequencing, and could not reflect the true expression of miRNA. S3 included 4 bases “ACGA” as a recognin. 1 μL RA5 adaptor (10 mM), 1 μL ATP (10 mM) and 1 μL T4 RNA Ligase were mixed, and added into the previous 11 μL reaction system at 28 oC for 1h, followed by 1 μL RNA RT Primer at 70 oC for 2 min. The system was further added 2 μL 5 × First Strand Buffer, 0.5 μL dNTP Mix (12.5 mM), 1 μL DTT (100 mM), 1 μL RNase Inhibitor, and 1 μL SuperScript Ⅱ Reverse Transcriptase at 50 oC for 1 h. The cDNA libraries were amplified by PCR for 15 cycles, and fragments of 150-200 nt were selected by electrophoresis and sequenced with single-end 75 as the sequencing mode on Illumina Novaseq 6000 platform.