characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-10-31T21:00:04Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-10-31T21:00:04Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112763661
characteristics.characteristic: External Id
characteristics.text: BIMSB
characteristics.characteristic: INSDC center name
characteristics.text: 2023-10-31T21:00:04Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-10-31T21:00:04Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14758360
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12757:TPSeq_HEK_WT_C_br3.fastq.gz
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: HEK293
characteristics.characteristic: cell_line
characteristics.text: human
characteristics.characteristic: common name
characteristics.text: Protocols: Subcellular fractionation, RNA extraction and 3’end sequencing library preparation. For 3’end sequencing cells were fractionated into cytoplasm and nuclei and RNA was extracted separately. 10 million HEK 293 cells were mixed with 1 million MEF cells as a spike-in control, washed once in 1xPBS and resuspended in 1 mL hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 1 mM DTT, 1 mM PMSF, 0.1% Triton, 1x protease inhibitors). Cells were incubated on ice for 5 min, then dounced in a prechilled 7 mL Douncer for 10 strokes with a tight pestle to release nuclei. After centrifugation at 228g, 4°C for 5 min the supernatant was mixed with 200 µL 5x RIPA buffer (250 mM Tris pH 7.5, 750 mM NaCl, 5% NP-40, 2.5% deoxycholate, 1 mM PMSF, 0.1% Triton, 1x protease inhibitors) and centrifuged for 10 min at 2800g, 4°C. The supernatant was kept as the cytoplasmic fraction. The pellet from the first centrifugation step was resuspended in 600 µL sucrose buffer 1 (0.25 M sucrose, 10 mM MgCl2, 1 mM PMSF, 1x protease inhibitors) and layered over 600 µL sucrose buffer 2 (0.35 M sucrose, 0.5 mM MgCl2, 1mM PMSF, 1x protease inhibitors). After centrifugation at 1430g for 5 min, 4°C, the pellet (nuclei) was resuspended in 1 mL TRI reagent and processed for RNA extraction as described above. For RNA extraction from the cytoplasmic fraction, 300 µL cytoplasmic extract were mixed with 900 µL TRI reagent LS for liquid samples (Sigma), 240 µL chloroform was added and the lysate was vortexed and centrifuged at max. speed (21130g) for 15 min at 4°C. The aqueous layer was transferred to a new tube and precipitated with 0.6 mL isopropanol. The RNA pellet was subsequently treated as described above. Library preparation for 3’ end sequencing of polyadenylated mRNAs was performed using 3’ mRNA-Seq Library Prep Kit FWD (Lexogen) according to the manufacturer’s instructions.
characteristics.characteristic: description
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: C-terminal localisation
characteristics.characteristic: phenotype
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: TPSeq_HEK_WT_C_br3.fastq.gz
characteristics.characteristic: title