GSM3408477: HMGB1_1; Homo sapiens; RNA-Seq
The HeLa cells were ground into fine powder before RNA extraction. Total RNA was extracted by the hot phenol method. The RNA was further purified with two phenol-chloroform treatments and then treated with RQ1 DNase (Promega, Madison, WI, USA) to remove DNA. The quality and quantity of the purified RNA were redetermined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad, USA). The integrity of RNA was further verified by 1.5% agarose gel electrophoresis. For each sample, 10μg of the total RNA was used for RNA-seq library preparation. Polyadenylated mRNAs were purified and concentrated with oligo(dT)-conjugated magnetic beads (Invitrogen, Carlsbad, CA, USA) before directional RNA-seq library preparation. The purified mRNAs were then iron fragmented at 95°C followed by end repair and 5' adaptor ligation. Then, reverse transcription was performed with RT primer harboring 3' adaptor sequence and randomized hexamer. The cDNAs were purified, amplified, and stored at -80°C until they were used for sequencing.  For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions. Illumina HiSeq 4000 was used to collect data from 150-bp pair-end sequencing (ABlife Inc., Wuhan, China).