characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-06-19T17:00:05Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-06-19T17:00:05Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA111539478
characteristics.characteristic: External Id
characteristics.text: BIMSB
characteristics.characteristic: INSDC center name
characteristics.text: 2023-06-19T17:00:05Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-06-19T17:00:05Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS13649542
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12359:TT_HEK_SHARP_WT_br2
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: In vitro transcribed synthetic spike-in RNA was prepared as described66. 1 µL ERCC RNA Spike-in Mix (Invitrogen) were reverse transcribed using Protoscript II reverse transcriptase (New England Biolabs). cDNA was diluted 1:4 in H2O and used as a template for PCR amplification of ERCC spike-ins 00043, 00170, 00136, 00145, 00092 and 00002. PCR products were purified and verified by agarose gel electrophoresis and Sanger sequencing. 0.5 µg PCR product were used as an input for in vitro transcription using the MEGAscript T7 transcription kit (Invitrogen) according to the manufacturer’s instructions. For spike-ins 00043, 00136 and 00092 10% UTP were substituted with 4-Thio-UTP. RNA spike-ins were purified using AMPure XP beads (Beckman Coulter), quantified using Qubit HS RNA Assay kit (Invitrogen) and mixed at a final concentration of 1 ng/µL of each spike-in RNA. The final spike-in mix was quantified again using Qubit HS RNA Assay kit (Invitrogen) and stored at -80 °C in single-use aliquots. For preparation of 4TU-labeled yeast RNA, an ON culture of S. cerevisiae BY4741 was diluted to OD600=0.1 in 50 mL YPD + 2% (w/v) glucose at grown at 30°C, 180 rpm until OD600=0.8. 4TU was added to a final concentration of 5 mM and cells were labeled for 5 min. Labeled cells were pelleted, resuspended in 250 µL 0.8 M sorbitol, 0.1 M EDTA, 0.1% β-mercaptoethanol, 200 U/mL lyticase and incubated at 30 °C for 30 min. Lysate was mixed with 750 µL TRI Reagent LS (Sigma), 200 µL Chloroform (Applichem) were added, samples were mixed and centrifuged at 4°C, max. speed for 15 min. The aqueous phase was subjected to isopropanol precipitation. RNA was treated with DNase I (Roche) at 37°C for 30 min and purified by phenol-chloroform extraction and Ethanol precipitation. Sequencing libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions.
characteristics.characteristic: description
characteristics.text: embryo stage
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: SHARP wild type
characteristics.characteristic: genotype
characteristics.text: not applicable
characteristics.characteristic: isolate
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: TT_HEK_SHARP_WT_br2
characteristics.characteristic: title