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| Status |
Public on Oct 24, 2022 |
| Title |
methylated_E. coli tRNA_WTTadA |
| Sample type |
SRA |
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| Source name |
methylated_E. coli tRNA_WTTadA
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| Organism |
Escherichia coli |
| Characteristics |
cell type: whole strain
treatment: WTTadA
library type: in vitro transcribed RNA
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| Extracted molecule |
total RNA |
| Extraction protocol |
Double-stranded DNA templates carrying T7 promoter were prepared by primer extension with two single-stranded DNA oligos. Unmethylated and methylated E. coli tRNA (Arg2, CGT), RNA#1, and RNA#2 were synthesized by in vitro transcription using T7 RNA polymerase. ATP and N6-methyl-ATP (TriLink, N-1013) were supplied in the presence of UTP, CTP, and GTP to synthesize unmethylated and methylated RNA, respectively. RNA was purified by E.Z.N.A Micro RNA kits (Omega Bio-Tek, R7034) and quantified by NanoDrop One (Thermo Fisher Scientific). All reactions were carried out in deamination buffer (50 mM Tris, 25 mM KCl, 2.5 mM MgCl2, 2 mM dithiothreitol, and 10 % (v/v) glycerol; pH 7.5) in the presence of 10 U SUPERaseIn RNase Inhibitor (Thermo Fisher Scientific, AM2694). RNA was always preheated to 95oC for 3 min and immediately cooled down before use. To assay deaminase activity on the natural substrate E. coli tRNA, 200 ng RNA and 100 nM wild type TadA or TadA8.20 were incubated at 37oC for 1 h. For a typical deamination assay on RNA probes, 10 ng RNA was incubated with 10 uM TadA8.20 in 20 uL deamination buffer at 37°C for 3 h. All reactions were quenched by incubating at 95°C for 10 min. Temperature and pH were adjusted to identify the optimal condition for in vitro deamination.
Both rounds of PCR were set up using EvaGreen qPCR Master Mix. 1st round PCR was carried out at a 20 uL scale with cDNA generated from different amounts of mRNA or total RNA, and 0.5 uM forward and reverse primers. Primers were designed to recognize sequences post deamination, leaving out 10-20 nt sequences surrounding the target m6A sites. PCR reactions were analyzed by agarose gel electrophoresis. 1 uL of PCR products were subjected to enzymatic cleanup (Exonuclease I and Shrimp Alkaline Phosphatase, New England BioLabs, M0293 and M0371) and Sanger sequencing. Gel purification was performed for PCR reactions that did not yield bright and single bands. 2nd round PCR was carried out at a 20 uL scale with 1 uL of the 1st round PCR reaction as template, 0.5 uM specified Illumina P7 and P5 index primer pairs. Barcoded PCR reactions were combined and gel purified using QIAquick Gel Extraction Kit (Qiagen, 28706) before being subjected to next-generation sequencing on an Illumina MiSeq Instrument.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
methylated_E. coli tRNA_WTTadA
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| Data processing |
For site-specific m6A quantification by amplicon deep sequencing, raw next-generation sequencing data were merged by Pear v0.9.8. before mapping to the target sequences using bwa-mem2 v2.2.1. Mapped reads were sorted and indexed using samtools v1.14. Bases mapped to individual A sites were counted using pysamstats v1.1.1. A and G counts were extracted for individual A sites covered by R2 using re.finditer. Methylation levels are reported as apparent A fractions. For site-specific m6A quantification by eTAM-Sanger, the A and G fractions observed at all A sites were quantified by EditR. Methylation levels are reported as apparent A fractions.
Assembly: NA
Supplementary files format and content: two synthetic RNA probes and one E. coli tRNA with site specific m6A quantificaton in the XLSX format
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| Submission date |
Apr 19, 2022 |
| Last update date |
Oct 24, 2022 |
| Contact name |
Shun Liu |
| Organization name |
University of Chicago
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| Department |
Chemistry
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| Lab |
Chuan He Lab
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| Street address |
929 E. 57th. Street
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL16085 |
| Series (2) |
| GSE201062 |
Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination [probes_assay] |
| GSE201064 |
Transcriptome-wide profiling and quantification of N6-methyladenosine by enzyme-assisted adenosine deamination |
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| Relations |
| BioSample |
SAMN27642101 |
| SRA |
SRX14918188 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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