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| Status |
Public on Sep 24, 2019 |
| Title |
Evolution of a Reverse Transcriptase to Map N1-Methyladenosine in Human mRNA |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
Chemical modifications on mRNA are increasingly recognized as a critical regulatory layer of the flow of genetic information, but quantitative tools to monitor RNA modifications in a whole-transcriptome and site-specific manner are lacking. Here we describe a versatile directed evolution platform that rapidly selects for reverse transcriptases that install mutations during reverse transcription at sites of a given type of RNA modification, allowing for site-specific identification of the modification. To develop and validate the platform, we evolved the HIV-1 reverse transcriptase against N1-methyladenosine (m1A). Iterative rounds of selection yielded reverse transcriptases with both robust read-through and high mutation rates at m1A sites. We apply the evolved reverse transcriptase to identify thousands of statistically confident m1A sites in human mRNA, some of which can be detected in antibody-free RNA-seq libraries. Together, this work develops and validates the reverse transcriptase evolution platform and provides new tools, analysis methods, and datasets to study m1A biology.
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| Overall design |
m1A-IP-seq and m1A-quant-seq experiments were performed on mRNA extracted from HEK-293T cells with three biological replicates.
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| Contributor(s) |
Zhou H, Dickinson BC, He C |
| Citation(s) |
31548705 |
| Submission date |
Dec 05, 2018 |
| Last update date |
Sep 25, 2019 |
| Contact name |
Huiqing Zhou |
| E-mail(s) |
zhouhq@uchicago.edu
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| Phone |
7343899318
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| Organization name |
University of chicago
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| Street address |
LOADING DOCK BEHIND 5741 S DREXEL AVE 929 E 57TH ST
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| City |
Chicago |
| State/province |
Illinois |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (12)
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| GSM3896228 |
m1A-quant-seq, "+AlkB", replicate1 |
| GSM3896229 |
m1A-quant-seq, "+AlkB", replicate2 |
| GSM3896230 |
m1A-quant-seq, "+AlkB", replicate3 |
| GSM3896231 |
m1A-IP-seq, "-AlkB", replicate1 |
| GSM3896232 |
m1A-IP-seq, "-AlkB", replicate2 |
| GSM3896233 |
m1A-IP-seq, "-AlkB", replicate3 |
| GSM3896234 |
m1A-IP-seq, "+AlkB", replicate1 |
| GSM3896235 |
m1A-IP-seq, "+AlkB", replicate2 |
| GSM3896236 |
m1A-IP-seq, "+AlkB", replicate3 |
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| Relations |
| BioProject |
PRJNA508500 |
| SRA |
SRP172548 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE123365_RAW.tar |
124.4 Mb |
(http)(custom) |
TAR (of CSV) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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