Illumina HiSeq 2000 paired end sequencing; The transcriptome-wide CPSF6-induced APA alterations in gastric cancer cells
The transcriptome-wide CPSF6-induced APA alterations in gastric cancer cells
Human normal GC cell line GES1 and GC cancer cell line AGS were purchased from the Chinese Academy of Sciences cell bank (Shanghai, China). All cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), penicillin (50 U/ml, Sangon, China), streptomycin (50 mg/ml, Sangon, China), at 37°C in a 5% CO2 humidified atmosphere. The lentiviral vector pLKO.1-puro-shRNA-CPSF6 was constructed for CPSF6 knockdown. 293T cells were transfected for the lentiviral production. After 48 h and 72 h, the supernatant was collected. Then AGS cells were infected by supernatant for 72 h. Finally, cells were maintained in the presence of puromycin (2mg/ml) for 1-2 weeks. Total RNA was extracted using TRIzol (Invitrogen, USA) according to the manufacturer's instructions. Briefly, 50 μg total RNA was incubated with M280 beads, which were bound by Bio-oligo dT (20). After cDNA synthesis, the 3′UTR fragments were released by Gsu I digestion and then subjected to deep sequencing.The libraries were constructed, using NEBNext® Ultra™ II DNA Library Prep Kit and following manufacturer's instructions.