characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2022-05-14T00:14:31Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2022-05-14T00:14:31Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA13127420
characteristics.characteristic: External Id
characteristics.text: The Francis Crick Institute
characteristics.characteristic: INSDC center name
characteristics.text: 2022-05-14T00:14:31Z
characteristics.characteristic: INSDC first public
characteristics.text: 2022-05-14T00:14:31Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS10731375
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-106891645546026:polysome_RCC4_noVHL_EIF4E2_NA_4_Torin1_ribo0B
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: RCC4
characteristics.characteristic: cell line
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: 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.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: clear cell renal carcinoma
characteristics.characteristic: disease
characteristics.text: ribopolysome fraction 0B
characteristics.characteristic: fraction
characteristics.text: empty vector
characteristics.characteristic: genotype
characteristics.text: clone 4
characteristics.characteristic: individual
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: kidney
characteristics.characteristic: organism part
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: polysome_RCC4_noVHL_EIF4E2_NA_4_Torin1_ribo0B
characteristics.characteristic: title