GSM7989671: si-NC2; Homo sapiens; RNA-Seq
Sample RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, obtaining 1.5 μg of RNA separately for each sample. The purity and concentration of RNA was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. Once the total RNA sample is up to standard, it is enriched with Oligo (dT) beads. Then the purified mRNA was fragmented into short fragments using fragmentation buffer. The first strand of cDNA was synthesized in the M-MuLV reverse transcriptase system using fragmented mRNA as a template and random oligonucleotides as primers. Add buffer, dNTPs, Rnase H and DNA Polymerase I to synthesize the second strand of cDNA. The purified double-stranded cDNA fragments were end repaired, A base added, and ligated to Illumina sequencing adapters. The ligation reaction was purified with the AMPure XP Beads (1.0X). Ligated fragments were subjected to size selection by agarose gel electrophoresis and polymerase chain reaction (PCR) amplified.The resulting cDNA library was sequenced using Illumina Novaseq6000 by Gene Denovo Biotechnology Co. (Guangzhou, China).