JP26
Homo sapiens
Protocols: HEK pTRMT10C cells and the control cells (Ctl) were seeded two days prior to the RNA isolation, each in three wells to test three different tetracycline concentrations. The next day, the media was replaced with growth medium, containing 0 µg/mL, 0.1 µg/mL and 1 µg/mL tetracycline. After incubation for 24 hours at 37°C in a humidified incubator containing 5% CO2, RNA isolation was performed. pTRMT10C cells: T-Rex-293 (Invitrogen, R71007) (Ctl cells) were transfected with a pcDNA™5/TO plasmid coding for full-length TRMT10C in frame with a FLAG-tag. The combination of T-Rex-293 and the TRTM10C plasmid enables a tetracycline-inducible overexpression of TRMT10C in dependece of the tetracycline concentration. Control T-Rex-293 (Ctl) cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, 50 Units/mL penicillin, 50 µg/mL streptomycin and 5 µg/mL blasticidin (Thermo Fisher Scientific, Cat.-No: R21001). Medium of pTRMT10C cells additionally contained 100 µg/mL hygromycin (Invitrogen, 10687010) for selecting the pcDNA™5/TO plasmid. 24 hours before the isolation of total RNA pTRMT10C as well as T-Rex-293 cells were incubated with 0 µg/mL, 0,1 µg/mL and 1 µg/mL tetracycline (Sigma-Aldrich, T7660). 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 was 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, the 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
2023-07-24T00:21:31Z
ENA-LAST-UPDATE
2023-07-24T00:21:31Z
External Id
SAMEA110472456
INSDC center alias
JOHANNES GUTENBERG UNIVERSITY MAINZ
INSDC center name
JOHANNES GUTENBERG UNIVERSITY MAINZ
INSDC first public
2023-07-24T00:21:31Z
INSDC last update
2023-07-24T00:21:31Z
INSDC status
public
Submitter Id
E-MTAB-12038:JP26
broker name
ArrayExpress
cell_line
HEK293-T-REx cell
cell_type
epithelial cell
common name
human
developmental stage
embryo stage
genotype
tetracycline-inducible TRMT10C expressing
isolate
not applicable
organism part
kidney
sample name
E-MTAB-12038:JP26
scientific_name
Homo sapiens