description: Protocols: Two VHL-defective kidney cancer cell lines, RCC4 and 786-O, were used. Sublines of these cells with reintroduction of VHL or an empty vector control were prepared using lentiviral transduction, whereas HIF1B was inactivated using CRISPR/Cas9 mediated genome editing. Cells were grown in DMEM with 1 mM sodium pyruvate, 10% FBS and 5 µg/mL blasticidin at 37 ˚C in 5% CO2. For analysis of the effect of hypoxia, cells were cultured at 37 ˚C in 1% O2 and 5% CO2 for 24 hours. An RNeasy Plus Mini Kit (QIAGEN, 74136) was used to extract RNA according to the manufacturer's instructions with the exception of technical replicate 2 of the samples from RCC4 cells. For these samples, cells were lysed with Buffer RLT Plus (QIAGEN, 1053393), and total RNA was extracted using an RNA clean and concentrator-25 kit (Zymo Research, R1018) according to the manufacturer's instructions, except that 752.5 µL of preconditioned RNA binding buffer (367.5 µL of RNA binding buffer (supplied with RNA clean and concentrator-25 kit), 367.5 µL of absolute EtOH and 17.5 µL of 20% SDS) was added to 350 µL of the cell lysate before loading onto the column. 5′ end-Seq was performed on the extracted total RNA. In short, a full-length cDNA library was generated using a combined reverse transcription and template switching reaction, followed by PCR amplification. The amplified libraries were multiplexed, and then purified using ProNex beads (Promega). The 3′ end of the cDNA library (i.e. 5′ end of mRNAs) was amplified specifically using Tn5 transposase-mediated tagmentation and PCR amplification with primers annealing to the appropriate adapters added by the template switching reaction and tagmentation. The PCR primers contained i5 and i7 Illumina index sequences and the amplified libraries were multiplexed.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2022-05-14
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2022-05-14
attributes.tag: External Id
attributes.value: SAMEA9372083
attributes.tag: INSDC center alias
attributes.value: The Francis Crick Institute
attributes.tag: INSDC center name
attributes.value: The Francis Crick Institute
attributes.tag: INSDC first public
attributes.value: 2022-05-14T00:14:05Z
attributes.tag: INSDC last update
attributes.value: 2022-05-14T00:14:05Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10688:total_RCC4_noVHL_HIF1B_H_1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell_line
attributes.value: RCC4
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: clear cell renal carcinoma
attributes.tag: genotype
attributes.value: empty vector
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: kidney
attributes.tag: sample name
attributes.value: E-MTAB-10688:total_RCC4_noVHL_HIF1B_H_1