The RNA-seq (miRNA and mRNA) analysis of three human osteosarcoma cells

RNA-seq analysis was performed by BGI-Tech of China, and RNA-seq library preparation and sequencing were performed by BGI (Shenzhen, China). Following purification, the RNA was fragmented using divalent cations at an elevated temperature, and first-strand cDNA was synthesized using random hexamer primers and Superscript TMIII (Invitrogen™, Carlsbad, CA, USA). Second-strand cDNA was synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. Short fragments were purified with a QiaQuick PCR extraction kit (Qiagen) and resolved with EB buffer for end repair and poly (A) addition. The short fragments were subsequently connected using sequencing adapters. After agarose gel electrophoresis, suitable fragments were used as templates for PCR amplification. During the QC steps, an Agilent 2100 Bioanalyzer and an ABI StepOnePlus Real-Time PCR System were used for the quantification and qualification of the sample library. Finally, the library (200 bp insert) was sequenced using an Illumina HiSeq 2000 (Illumina Inc., San Diego, CA, USA). The single-end library was prepared following the protocol of the Illumina TruSeq RNA Sample Preparation Kit (Illumina)