title: GSM9251915: SW480 cells, pCMV6-HKDC1_2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted using Tri Reagent (Ambion, Austin, TX, USA), and the RNA concentration and quality were evaluated using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA).  Total RNA with A260/A280 = 1.8-2.0 and RNA integrity number >8.0, were used for further experiments. Total RNA (1–2 μg) was purified using poly-T oligo–attached magnetic beads to isolate mRNA. The purified mRNA was fragmented into small pieces using divalent cations at elevated temperature. First-strand cDNA was synthesized using reverse transcriptase and random primers, followed by second-strand cDNA synthesis with DNA polymerase I and RNaseH. The resulting cDNA fragments underwent end repair, addition of a single adenine (“A”) base at the 3′ ends, and ligation of sequencing adapters. Finally, the adapter-ligated products were purified and enriched by PCR to generate the sequencing-ready cDNA library.