Illumina NovaSeq 6000 paired end sequencing; RNA-seq of a VHL-/- human renal cancer cell line 786-0 transfected with 3 vectors
The three cell lines were maintained in culture, treated with 1 g/mL doxycycline for two weeks to allow VHL expression. Then, total RNA extraction, RNA integrity, RNA sequencing and analysis  were performed.Each sample was done in triplicate. The three cell lines were maintained in culture in DMEM medium (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA ), supplemented with 5% tetracycline-free fetal calf serum (FCS) (PAA Laboratories Inc, Ontario, Canada), 1% sodium pyruvate, 1% penicillin/streptomycin, 0.2 mg/mL zeocin, 3 g/mL blasticidin.  The three cells lines were treated with 1 g/mL doxycycline for two weeks to allow VHL expression.  Total RNA extraction was performed with an RNA/DNA purification kit (Norgen, UK).  dsDNA libraries were generated from the fragmented mRNAs, bar-coded and purified.