2.5 RNA extraction and sequencing
The HeLa cells were triturated into fine powder before RNA extracted with TRIZOL (Ambion). The RNA was further refined with two phenol-chloroform treatments and then preformed with RQ1 DNase (Promega, Madison, WI, USA) to remove DNA. The quality and quantity of the refined RNA were rejudged by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad, USA). The integrity of RNA was further determined by 1.5% agarose gel electrophoresis.
For each sample, 1μg of the total RNA was utilized for RNA-seq library preparation by VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme). Polyadenylated mRNAs were refined and fragmented, and then translated into double strand cDNA. After the step of end repair and A tailing, the DNAs were roped to VAHTS RNA Adapters (Vazyme). Refined ligation products corresponding to 200-500 bps were digested with heat-labile UDG, and before sequencing ,the single strand cDNA was amplified, refined, quantified and stored at -80℃.
The libraries were prepared following the manufacturer’s instructions and applied to Illumina HiSeq X Ten system for 150 nt paired-end sequencing for high-throughput sequencing.