GSM7324744: USF1_2nd; Homo sapiens; RNA-Seq
The collected Huh7 cells were subjected to total RNA extraction using TRIZOL (15596-018, Ambion, USA).  The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1 DNase (M6101, Promega, Madison, WI, USA) to remove DNA.  The quality and quantity of the purified RNA were determined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Ultrafine spectrophotometer (N50 touch, IMPLEN, Germany).  The integrity of RNA was further verified by 1.5% agarose gel electrophoresis. For each sample, 1μg of the total RNA was used for RNA-seq library preparation by VAHTS Stranded mRNA-seq Library Prep Kit (NR605-02, Vazyme, China). Polyadenylated mRNAs were purified and fragmented, and then converted into double strand cDNA. After the step of end repair and A tailing, the DNAs were ligated to VAHTS RNA Adapters. Purified ligation products corresponding to 200-500 bps were digested with heat-labile UDG, and the single strand cDNA was amplified, purified, quantified and stored at -80℃ before sequencing. For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina Nova6000 system for 150 nt paired-end sequencing.