Illumina HiSeq 2000 paired end sequencing; The transcriptome-wide CPSF6-induced APA alterations in HCC cells
The transcriptome-wide CPSF6-induced APA alterations in HCC cells
Human immortalized liver cell line (HL-7702 (L-02)) and liver cancer cell lines Huh-7 were purchased from Shanghai Cell Bank (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Carlsbad, CA, USA) supplemented with 10% feta bovine serum (FBS, Gibco, Carlsbad, CA, USA), penicillin (50 U/ml, Sangon, Shanghai, China), streptomycin (50 mg/ml, Sangon, Shanghai, China), and glutamine (2mM, Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 humidified atmosphere. The lentiviral vectors pSIN-GFP-puro-CPSF6 was constructed for CPSF6 overexpression. The lentiviral vectors pLKO.1-puro-shRNA-CPSF6 was constructed for CPSF6 knockdown. The 293T cell was used for the lentiviral production and supernatant was collected at 48 and 72 h after transfection. The viruses were filtered using 0.45 micrometers filters and appropriate amounts of viruses were used to infect liver cells. Finally, cells were maintained in the presence of puromycin (2μg/mL) for 1–2 weeks. Total RNA was extracted using TRIzol (Invitrogen,Carlsbad, CA, USA) according to the manufacturer's instructions. 3T-seq library preparation was performed as we described previously. Briefly, M280 beads was coated with Bio-oligo dT(20) and then incubated with 50 micrograms total RNA. After that the cDNA was synthesized and released by GsuI digestion and then the 3′UTR fragments were ligated with Illumina sequencing adapters.
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University
E-MTAB-9458:HL7702_3T-seq_control_p