HEK ut cells, Total RNA used for RT-PCR, biological replicate 2
Homo sapiens
Protocols: HEK 293 cells were transfected with a DNA construct harboring the human APPwt gene, inserted downstream of a cytomegalovirus promoter using the FUGENE 6 technology (Roche Diagnostics). The stably expressing APPwt HEK 293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco, REF: 41965-039) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Gibco, REF: 10270-106, Lot: 2156880), 50 Units/mL penicillin and 50 µg/mL streptomycin (Pen Strep, Gibco, REF: 15140-122), and 400 µg/mL G418 Solution (Roche, 4727894001) at 37 °C in a humidified incubator containing 5% CO2. Control cells (Ctl) (untransfected HEK 293) were cultured in the same medium, excluding G418. Thus, a mitochondria isolation was done in order to enrich the desired mitochondrial mRNA. Therefor HEK cells were washed with ice-cold PBS and seeded in large cell culture plates. After two days growing, medium was changed and on the third day the isolation of mitochondria was performed based on a Percollgradient. Cells were seeded two days in advance and washed with ice-cold PBS for synchronization. Cells were harvested at a confluency of 70-80% in 1 mL TRI Reagent®. Dissolution of the cells was supported by vortexing and incubation at room temperature for 5 min. Next 200 µL of chloroform (Sigma-Aldrich, C2432) was added and mixed thoroughly. After a second incubation step (10 min) at 21 °C, the cell suspension was centrifuged (16 000 x g, 4 °C, 15 min). The upper aqueous phase was taken and mixed in a 1:1 ratio with isopropanol (ROTH, 7343.1) and 1 µL glycogen (Thermo Scientific, R0551) was added, incubated at 21 °C (5 min) and centrifuged (16 000 x g, 4 °C, 10 min). The RNA pellet was washed twice with ice-cold 75% ethanol including centrifugation (16 000 x g, 4 °C, 5 min). RNA was reconstituted in 10-20 µL nuclease-free water (Invitrogen, AM9930). RNA concentrations were determined using UV-VIS spectrophotometer Nanodrop 2000 (Thermo Fisher Scientific, Waltham, USA). Total RNA and RNA from mitochondrial extracts were incubated with DNase I (Zymo Research, E1010) and 10x DNase digestion Buffer (Zymo Research, E1010) at 21 °C (15 min) to eliminate any existing DNA contamination and a second phenol-chloroform extraction was performed as described above. For reverse transcription 2 µg RNA, 0.5 mM dNTPs (Thermo Scientific, R0192), water and a target-specific RT primer (see Supplementary Table S1) (2.5 µM) were mixed and incubated for 5 min at 75 °C and afterwards 5 min on ice. Next 10 U/µL SuperScript-IV (Invitrogen, 18090050), dithiothreitol (DTT) and 5x RT Buffer and were added, mixed and incubated for 2 hours at 50 °C. In the end, enzyme was inactivated at 80 °C for 15 min. The RT Primer contained a target-specific sequence complementary to ND5 mRNA and a primer-binding site for indexing with the NEBNext® Multiplex Small RNA Library Prep Kit for Illumina® (New England Biolabs, #E7560S) primers afterwards during polymerase chain reaction (PCR). PCR was conducted with 0.04 U/µL Platinum SuperFi (Invitrogen, 12351010), 5x SuperFi buffer (Invitrogen, 12351010), 0.2 mM dNTPs, 5x GC Enhancer (Invitrogen, 12351010), 0.5 µM custom-designed forward primer (see Supplementary Table S1) and reverse primers of the NEBNext® Multiplex Small RNA Library Prep Kit for Illumina®. The custom p5 PCR primer contained flow cell binding sequence, sequencing primer site 1 and a target-specific sequence complementary to ND5 cDNA. The commercial i7 index primers introduced individual barcodes for each sample, contained the sequencing primer site 2 and the second flow cell binding sequence. PCR was started in Thermocycler peqSTAR (VWR Peqlab), including an initial denaturation step at 98 °C (2 min), 30 cycles at 98 °C (15 min), 63 °C (1 min) and 72 °C (2 min), and a final extension step at 72°C (5 min). PCR products were purified on a 10% denaturing polyacrylamide gel by size selection and the expected full-length product was excised. Finally, prepared libraries were quality controlled for adapter dimer formation on an Agilent 4200 TapeStation (Agilent, Santa Clara, United States).
ENA-FIRST-PUBLIC
2023-07-22T00:53:09Z
ENA-LAST-UPDATE
2023-07-22T00:53:09Z
External Id
SAMEA110464431
INSDC center alias
JOHANNES GUTENBERG UNIVERSITY MAINZ
INSDC center name
JOHANNES GUTENBERG UNIVERSITY MAINZ
INSDC first public
2023-07-22T00:53:09Z
INSDC last update
2023-07-22T00:53:09Z
INSDC status
public
Submitter Id
E-MTAB-12033:HEK ut cells, Total RNA used for RT-PCR, biological replicate 2
broker name
ArrayExpress
cell_line
HEK293
common name
human
developmental stage
embryo
genotype
wild type genotype
isolate
not applicable
organism part
kidney
phenotype
wild type
sample name
E-MTAB-12033:HEK ut cells, Total RNA used for RT-PCR, biological replicate 2
scientific_name
Homo sapiens