GSM7506630: Tumor_LN_2; Homo sapiens; ncRNA-Seq
Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manual instructions. Total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Then, 3μg of total RNA was treated with DNase I which degraded double-stranded and single-stranded DNA presenting in RNA samples. And the ribosomal RNA was removed using the Ribo-off rRNA Depletion Kit (Vazyme, Inc.). Linear RNA was removed using RNase R (Epicentre, lnc). Purification was performed using Agencourt RNAClean XP magnetic beads. All other steps were performed according to the manufacturer's protocols. The library was quality and quantitated in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using BMG (OMEGA). RNA libraries were prepared for sequencing using standard DNBSEQ protocols RNase R degrades the abundant linear RNAs. Then, each sample was amplified and transcribed into fluorescent cRNA utilizing a random priming method. Double-stranded cDNA fragments are subjected to end-repair and A-tailing. After amplification by PCR, the reaction system and program for circularization are configured and set up. The concentration and specific activity of circRNAs were measured by DNBSEQ-G400.