Cells were transfected with 20 nM siRNA targeting RIOK3 (SI02223396; siRIOK3) or AllStars Negative control (1027281; siCon) using HiPerFect reagent (Qiagen, Manchester, UK). Custom siRNA oligonucleotides containing 3 sequences targeting HIF-1α (1 CAAGCAACTGTCATATATA, 2 TGCCACCACTGATGAATTA, 3 TGACTCAGCTATTCACCAA) or HIF-2α (1 TAACGACCTGAAGATTGAA, 2 CAAGCCACTGAGCGCAAAT, 3 TGAATTCTACCATGCGCTA) were purchased from Eurogentec (Southampton, UK). pGIPZ plasmids targeting RIOK3 (Clone ID V2LHS_16875; shRIOK3) or non-silencing control shRNAmir (RHS4743; shCon) were obtained from Thermo Scientific. Lentivirus packaging and titration was carried out as described.38 (link) Cells were transduced at MOI 1 and pools of transduced cells were selected with 1 μg/mL puromycin (Life Technologies) 48 h post-transduction.
Protocol detail
Silencing RIOK3 and HIF Transcription
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Corresponding Organization :
Other organizations : John Radcliffe Hospital, University of Oxford, Karolinska Institutet, Linköping University, University of Leicester
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Variable analysis
independent variables
- SiRNA targeting RIOK3 (SI02223396; siRIOK3)
- AllStars Negative control (1027281; siCon)
- Custom siRNA oligonucleotides targeting HIF-1α
- Custom siRNA oligonucleotides targeting HIF-2α
- PGIPZ plasmids targeting RIOK3 (Clone ID V2LHS_16875; shRIOK3)
- Non-silencing control shRNAmir (RHS4743; shCon)
- Unspecified
- HiPerFect reagent (Qiagen, Manchester, UK) used for transfection
- Puromycin (Life Technologies) used for selection of transduced cells
- Negative control: AllStars Negative control (1027281; siCon)
- Negative control: Non-silencing control shRNAmir (RHS4743; shCon)
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