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| Status |
Public on Mar 15, 2023 |
| Title |
Ψ-modified RNA oligo used in screening - RT-KTq - 100µMdATP |
| Sample type |
SRA |
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| Source name |
chemically synthesized oligo
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| Organism |
synthetic construct |
| Characteristics |
protocol: Ψ-modified RNA; RT by RT-KTq with 100 µM dNTP
molecule type: 53 nt RNA oligonucleotide
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| Growth protocol |
The S. cerevisiae strains were grown in standard YPD medium (20 g/L peptone, 20 g/L dextrose, 10 g/L yeast extract) at 30°C to exponential phase (0.6-0.7 OD600)
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| Extracted molecule |
other |
| Extraction protocol |
Total RNA from yeast cells was isolated using hot acid phenol. RNA concentration was measured on Nanodrop One and RNA quality was assessed by capillary electrophoresis using a PicoRNA Chip on Bioanalyzer 2100 (Agilent technologies). Yeast total tRNA fraction was enriched using NucleoBond RNA 80 kit (Macherey-Nagel). Briefly, 50-80 micrograms of total RNA were loaded onto AXR80 column previously equilibrated with RO buffer. The column was washed 4 times with R1 buffer and tRNA fraction was eluted using R2 buffer. The eluate was then precipitated with an equal volume of isopropanol and centrifuged. The pellet was washed with 80% ethanol, and dissolved with RNAse free water
For RNA oligo samples: RT-Reaction mix contained 225 nM RNA template (unmodified or modified oligonucleotide), 150 nM DNA reverse primer with overhang, 100 µM of each nucleotide and 2 nM RT-KTq variant in 1x RT-KTq reaction buffer (50 mM Tris-HCl (pH 9.2), 16 mM (NH4)2SO4, 25 mM MgCl2, 0.1% (v/v) Tween 20). After annealing of template/primer and heating the mixture to 55°C, 4 µL DNA polymerase in 1x RT-KTq reaction buffer was added to start reverse transcription (10 µL reaction volume). After 10 min reaction time, mixture was heated to 95°C for 5 min and annealing was allowed once again by cooling stepwise to 4°C. The reaction was heated to 55°C and 10 µL Superscript IV reaction mix (Invitrogen) was added to reach a final volume of 20 µL. Reactions contained finally 10 U/µL Superscript IV (Invitrogen), 300 µM dNTP each, 5 mM DTT and 1x Superscript IV RT buffer (Invitrogen). After primer elongation for 10 min at 55°C, the reaction was put on ice. For each DNA library, the described reverse transcription was performed three times and combined after reaction. 60 µL combined reaction mix was mixed with 240 µL Buffer NTC (Macherey-Nagel) and clean-up was conducted with NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel) according to the manufacturer's specifications. Elution was achieved by adding 15 µL Milli-Q water and centrifugation for 1 min at 13.000 rpm. Then, RNA template was digested by addition of 0.05 U/µL RNase H (NEB) in 1x RNase H reaction buffer at 37°C for 20 min. Enzyme was inactivated by heat at 65°C for 20 min. Purification of DNA was performed by agarose gel electrophoresis (2.5 % agarose gel in 1x TAE buffer), excision of the respective bands and applying the NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel). To achieve DNA binding to the columns, buffer NTC was added to the gel pieces (400 µL buffer per 100 mg gel) and incubated at 50°C until gel was dissolved. Elution was conducted in the same way as described above. Afterwards, DNA was repaired by using 0.1 µL PreCR Repair mix (NEB) with 100 µM of each dNTP, 1x NAD+ (provided by the manufacturer) and 1x ThermoPol reaction buffer (NEB) to reach 20 µL reaction volume. DNA repair was performed at 37°C for 20 min. Without any purification step, DNA was used as template for primer extension reaction with 0.02 U/µL Q5 Hot Start High-Fidelity DNA Polymerase (NEB), 200 µM of each dNTP, 300 nM DNA forward primer with overhang and 0.2x Q5 reaction buffer in 25 µL reaction volume. A one step cycling protocol was chosen where denaturation at 98°C for 1 min, annealing at 62°C for 45 s and finale extension at 72°C for 2 min were conducted. Reaction was analyzed and purified by agarose gel electrophoresis as described above. This time NTI buffer (Machery Nagel) was used for dissolving the gel pieces at 55°C. A second DNA repair with the PreCR mix (NEB) was conducted in the similar way as described above. UMI introduction was obtained by applying Q5 Hot Start High-Fidelity DNA Polymerase (NEB) in a three step PCR reaction. The reaction mixture contained 20 µL repair mix, 0.02 U/µL DNA polymerase, 200 nM forward and reverse primer and finally 0.2x Q5 reaction buffer to reach 25 µL reaction volume. After initial denaturation at 98°C for 2 min, two cycles with 98°C for 10 s, 62°C for 30 s and 72°C for 30 s were carried out followed by a final elongation step at 72°C for 2 min. Reaction was analyzed and purified by agarose gel electrophoresis as described above. Here, buffer NTI was diluted with Milli-Q in a ratio of 2:1 and afterwards used for dissolving gel pieces at 50°C. Concentration of the DNA was determined with a qPCR approach, where an oligonucleotide with the same length was applied as reference in different template concentrations. 1x NEBNext Ultra II Q5 Master Mix (NEB) was used for PCR with 400 nM fwd and rev primer, 1x sybr green I in 5 µL reaction volume. After a final denaturation step at 98°C for 1 min, a three step cycling protocol with 35 PCR cycles followed with 98°C 10 s, 60°C 30 s and 72°C for 30 s and finally an elongation step with 72°C for 2 min was conducted. Fluorescent intensity was measured at the end of each cycle and melting curves were determined at the end of the reaction. For analysis, Cq values of reactions with reference DNA were blotted against the decadic logarithm of the template concentrations and linear regression was created. DNA concentrations were calculated by using the linear predictor function. Results were multiplied by two since reference DNA was applied double stranded and target DNA in the analyzed samples was only present in the single stranded format. DNA was repaired once again by using 0.05 µL PreCR mix (NEB), 1x NAD+, 100 µM of each dNTP, 59.044 fM DNA and 1x ThermoPol reaction buffer (NEB) to reach 11.25 µL end volume. After reaction for 20 min at 37°C, DNA mix was directly used for Amplicon PCR. Here, 400 nM forward and reverse primer (equipped with Illumina Adapter Sequence and Indices), the entire repair mix (final DNA concentration 26.57 fM) and 1x NEBNext Ultra II Q5 Master Mix (NEB) was used for reaction in 25 µL end volume. After a final denaturation step at 98 °C for 5 min, a three step cycling protocol with 35 PCR cycles followed with 98°C 10 s, 70°C 30 s and 72°C for 30 s and finally an elongation step with 72°C for 2 min was conducted. Reaction was analyzed and purified by agarose gel electrophoresis as described above. To dissolve gel pieces, buffer NTI (Macherey Nagel) was diluted with Milli-Q water in ration 2:1 and 450 µL NTI mix was added to the gel pieces. After complete dissolving at 50°C, mixture was dilute once again with Milli‑Q water to reach a final volume of 1750 µL. DNA was loaded on the column and eluted with 20 µL Milli-Q water. After performing the repair reaction on the whole eluate as described above, DNA was purified by using the QIAEX II System (Qiagen). 2 µL particle slurry was used for each sample and elution of DNA was achieved by adding 18 µL Milli-Q to the dried beads, incubation for 5 min and taking 16 µL after centrifugation. The concentration of the final DNA library was determined by Quantus™ Fluorometer (Promega) and usually ranged between 20 and 85 nM. After quality control with Bioanalyzer 2100 (Agilent), DNA libraries were pooled and sequenced in paired-end mode (2*75 bp) on an IIlumina NextSeq2000 instrument. For tRNA samples: 300 ng RNA (isolated from S. cerevisiae) was annealed to 300 nM reverse primer (containing UMI sequence) in the similar way as described in the chapter above. Hybridized template/primer was added to the reaction mix containing 100 µM of d(G/C/T)TP (each) and 2 µM dATP in 1x RT-KTq reaction buffer (50 mM Tris-HCl (pH 9.2), 16 mM (NH4)2SO4, 25 mM MgCl2, 0.1% (v/v) Tween 20). After heating to 55°C, RT was started by adding 100 nM RT-KTq I614Y in 1x RT-KTq reaction buffer to reach a final reaction volume of 10 µL. After 1 h reaction time, mixture was heated to 95°C for 5 min and annealing was allowed once again by cooling to 4°C stepwise. The reaction was heated to 55°C and 10 µL Superscript IV reaction mix was added to reach a final volume of 20 µL. Reactions contained finally 10 U/µL Superscript IV (Invitrogen), 300 µM dNTP each, 5 mM DTT and 1x Superscript IV RT buffer (Invitrogen). After primer elongation for 10 min at 55°C, the reaction was put on ice. Reaction mix was mixed with 160 µL Buffer NTC (Macherey-Nagel) and clean-up was conducted with NucleoSpin Gel and PCR clean‑up XS kit (Macherey-Nagel) according to the manufacturer's specifications. Elution was achieved by adding 16 µL elution buffer (Machery-Nagel). DNA was used for second UMI Introduction with 0.02 U/µL Q5 Hot Start High-Fidelity DNA Polymerase (NEB), 200 µM of each dNTP, 200 nM DNA forward primer and 1x Q5 reaction buffer in 25 µL reaction volume. A one step cycling protocol was chosen where denaturation at 98°C for 1 min, annealing at 62°C for 45 s and finale extension at 72°C for 2 min were conducted. Buffer NTI (Macherey-Nagel) was mixed with water in ratio of 3:1 and 50 µL NTI-mix was added to the PCR reaction mix. The NucleoSpin Gel and PCR clean‑up XS kit (Macherey-Nagel) was used for purification and 16 µL elution buffer (Macherey-Nagel) was added for elution. Concentration of the DNA was determined with a qPCR approach described in the chapter above. DNA was repaired by using 0.05 µL PreCR mix (NEB), 1x NAD+, 100 µM of each dNTP, 56.008 fM DNA and 1x ThermoPol reaction buffer (NEB) to reach 11.25 µL end volume. After reaction for 20 min at 37°C, DNA mix was directly used for Amplicon PCR with NEBNext Ultra II Q5 Master Mix (NEB) as described in the chapter above (final DNA concentration 22 fM). Purification of DNA was performed by agarose gel electrophoresis (2.5 % agarose gel in 1x TAE buffer), excision of the respective bands and applying the NucleoSpin Gel and PCR clean-up XS kit (Macherey-Nagel). Here, buffer NTI (Macherey-Nagel) was diluted with Milli-Q water in ration 2:1 and 450 µL NTI mix was added to the gel pieces. After complete dissolving at 50°C, mixture was dilute once again with Milli‑Q water to reach a final volume of 1750 µL. DNA was loaded on the column and eluted with 20 µL elution buffer (Macherey-Nagel). DNA Repair on the whole eluate and purification with the QIAEX II system (Qiagen) was performed as described in the chapter above. The concentration of the final DNA library was determined by Quantus™ Fluorometer (Promega) and usually ranged between 6 and 46 nM. After quality control with Bioanalyzer 2100 (Agilent), DNA libraries were pooled and sequenced in paired-end mode (2*75 bp) on an IIlumina NextSeq2000 instrument.
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Sequencing data were processed by using the open source software KNIME 4.6.2
Quality and sequence are read from the FastQ files
Read1 and Read 2 were merged giving the expected size of transcript (RNA oligo samples 109 bp & tRNA samples 69 bp)
High quality data were filtered and aligned to the RNA reference of the oligonucleotide or tRNA sequences (http://gtrnadb.ucsc.edu).
Reads were sorted into unique molecular identifier (UMI) families and family read numbers were counted.
Error calculation are performed in each UMI family having more than 10 or 15 (tRNA data) reads.
If 90 % of the reads within each family carry the misincorporation, the error is set to 1 (otherwise 0).
Afterwards, the error is calculated over all UMI families at each position of the transcript and plotted using Microsoft excel software
Supplementary files format and content: excel-files include position, reference RNA sequence and error rates
Library strategy: DNA Amplicon-Seq
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| Submission date |
Nov 03, 2022 |
| Last update date |
Mar 15, 2023 |
| Contact name |
Luisa Barbara Huber |
| E-mail(s) |
[email protected]
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| Organization name |
Universität Konstanz
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| Department |
Department of Chemistry
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| Lab |
Andreas Marx Lab
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| Street address |
Universitätsstraße 10
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| City |
Konstanz |
| State/province |
Baden-Württemberg |
| ZIP/Postal code |
78464 |
| Country |
Germany |
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| Platform ID |
GPL32628 |
| Series (1) |
| GSE217198 |
Detection of Ψ in RNA oligonucleotides and in tRNAs from S. cerevisiae strains by reverse transcription using RT-KTq I614Y |
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| Relations |
| BioSample |
SAMN31589863 |
| SRA |
SRX18157154 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6706481_AM50_Al12_Min10.xlsx |
10.5 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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