Small RNA-seq analysis of serum miRNAs from patients with systemic sclerosis, Raynaud's phenomenon, and healthy controls
Small RNA-seq analysis of serum miRNAs from patients with systemic sclerosis, Raynaud's phenomenon, and healthy controls
Peripheral blood samples were collected in clot activator gel tubes (BD, United Kingdom), allowed to clot at room temperature, and then centrifuged to obtain serum, which was aliquoted and stored at −80 °C until analysis. After two subsequent spins, total RNA, including small (< 200 nucleotides) RNAs, was extracted from serum using Norgen's total RNA Purification Kit (cat. no. 37500, Norgen Biotek Corporation, Canada) according to the manufacturer's protocol. Purified RNA was stored at −80°C until analysis. Small RNA sequencing libraries were prepared from 5 μl of plasma RNA using the QIAseq miRNA Library Kit (catalog no. 331505, Qiagen, Hilden, Germany) according to the manufacturer's instructions. The quality and concentration of libraries were determined using the High Sensitivity DNA ScreenTape Analysis on the TapeStation 4150 system (Agilent Technologies, Santa Clara, CA, USA). 