description: Protocols: The VHL-defective kidney cancer cell lines, RCC4 and 786-O, were used. These cell lines were modified either by lentiviral transduction (to re-introduce VHL or an empty vector as control) and/or by CRISPR/Cas9-mediated genome editing of the EIF4E2 locus. Cells were grown on 15 cm dishes in DMEM with 1 mM sodium pyruvate, 10% FBS and 5 µg/mL blasticidin at 37 ˚C in 5% CO2. mTOR activity was inhibited by treating the cells with 250 nM of Torin 1 for 2 hours. mRNA translation was arrested by treating the cells with 100 µg/mL cycloheximide for 3 min prior to harvesting the cells. Cells were washed with ice-cold PBS with 100 µg/mL cycloheximide, and lysed using cytoplasmic lysis buffer. The cytoplasmic lysate was fractionated according to the number of associated ribosomes using sucrose density gradients and ultracentrifugation. RNA from the polysome fractionated samples was extracted using an RNA clean and concentrator-5 kit (Zymo Research, R1016) according to the manufacturer's instructions, with the following modifications: 322.5 µL of preconditioned RNA binding buffer (157.5 µL of RNA binding buffer (supplied with RNA clean and concentrator-5 kit), 157.5 µL of absolute EtOH and 7.5 µL of 20% SDS) was added to 150 µL of the fractionated samples before loading to 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: SAMEA9362387
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: 2022-05-14T00:14:04Z
attributes.tag: INSDC last update
attributes.value: 2022-05-14T00:14:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-10689:polysome_RCC4_noVHL_EIF4E2_NA_4_Torin1_ribo1
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: fraction
attributes.value: ribopolysome fraction 1
attributes.tag: genotype
attributes.value: VHL lentivirus
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: kidney
attributes.tag: sample name
attributes.value: E-MTAB-10689:polysome_RCC4_noVHL_EIF4E2_NA_4_Torin1_ribo1
attributes.tag: stimulus
attributes.value: Torin1