title: Illumina HiSeq 2000 paired end sequencing; The transcriptome-wide CPSF6-induced APA alterations in gastric cancer cells
design: The transcriptome-wide CPSF6-induced APA alterations in gastric cancer cells
library_construction_protocol: 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.
attributes.tag: Experimental Factor: RNA interference
attributes.value: CPSF6 knockdown by siRNA