Protocol for base resolution mapping of ac4C using RedaC:T-seq

David Sturgill; Daniel Arango; Shalini Oberdoerffer

doi:10.1016/j.xpro.2022.101858

Protocol for base resolution mapping of ac4C using RedaC:T-seq

David Sturgill^*, Daniel Arango^*, Shalini Oberdoerffer^*

^*Corresponding author for this work

Pharmacology

Research output: Contribution to journal › Article › peer-review

8 Scopus citations

Abstract

N4-acetylcytidine (ac4C) is an mRNA modification catalyzed by the enzyme N-acetyltransferase 10 (NAT10), with position-dependent effects on mRNA translation. This protocol details a procedure to map ac4C at base resolution using NaBH₄-induced reduction of ac4C and conversion to thymidine followed by sequencing (RedaC:T-seq). Total RNA is ribodepleted and then treated with NaBH₄ to reduce ac4C to tetrahydro-ac4C, which specifically alters base pairing during cDNA synthesis, allowing the detection of ac4C at positions called as thymidine following Illumina sequencing. For complete details on the use and execution of this protocol, please refer to Arango et al. (2022).¹

Original language	English (US)
Article number	101858
Journal	STAR Protocols
Volume	3
Issue number	4
DOIs	https://doi.org/10.1016/j.xpro.2022.101858
State	Published - Dec 16 2022

Funding

This protocol utilized the Center for Cancer Research (CCR) Sequencing Facility for the National Cancer Institute (NCI, Frederick, MD) for Illumina sequencing services. This protocol used the Biowulf Linux cluster at the National Institutes of Health, Bethesda, MD ( https://hpc.nih.gov ) to analyze sequencing data. This work is supported by the Intramural Research Program of NIH , Center for Cancer Research , National Cancer Institute . D.A. was supported by the NCI K99/R00 grant R00CA245035 .

Keywords

Bioinformatics
Molecular Biology
RNAseq
Sequencing

ASJC Scopus subject areas

General Biochemistry, Genetics and Molecular Biology
General Immunology and Microbiology
General Neuroscience

Access to Document

10.1016/j.xpro.2022.101858

Cite this

@article{ddbbb85d24dc4cb89f9cb472cf38baeb,

title = "Protocol for base resolution mapping of ac4C using RedaC:T-seq",

abstract = "N4-acetylcytidine (ac4C) is an mRNA modification catalyzed by the enzyme N-acetyltransferase 10 (NAT10), with position-dependent effects on mRNA translation. This protocol details a procedure to map ac4C at base resolution using NaBH4-induced reduction of ac4C and conversion to thymidine followed by sequencing (RedaC:T-seq). Total RNA is ribodepleted and then treated with NaBH4 to reduce ac4C to tetrahydro-ac4C, which specifically alters base pairing during cDNA synthesis, allowing the detection of ac4C at positions called as thymidine following Illumina sequencing. For complete details on the use and execution of this protocol, please refer to Arango et al. (2022).1",

keywords = "Bioinformatics, Molecular Biology, RNAseq, Sequencing",

author = "David Sturgill and Daniel Arango and Shalini Oberdoerffer",

note = "Publisher Copyright: {\textcopyright} 2022",

year = "2022",

month = dec,

day = "16",

doi = "10.1016/j.xpro.2022.101858",

language = "English (US)",

volume = "3",

journal = "STAR Protocols",

issn = "2666-1667",

publisher = "Cell Press",

number = "4",

}

TY - JOUR

T1 - Protocol for base resolution mapping of ac4C using RedaC:T-seq

AU - Sturgill, David

AU - Arango, Daniel

AU - Oberdoerffer, Shalini

PY - 2022/12/16

Y1 - 2022/12/16

N2 - N4-acetylcytidine (ac4C) is an mRNA modification catalyzed by the enzyme N-acetyltransferase 10 (NAT10), with position-dependent effects on mRNA translation. This protocol details a procedure to map ac4C at base resolution using NaBH4-induced reduction of ac4C and conversion to thymidine followed by sequencing (RedaC:T-seq). Total RNA is ribodepleted and then treated with NaBH4 to reduce ac4C to tetrahydro-ac4C, which specifically alters base pairing during cDNA synthesis, allowing the detection of ac4C at positions called as thymidine following Illumina sequencing. For complete details on the use and execution of this protocol, please refer to Arango et al. (2022).1

AB - N4-acetylcytidine (ac4C) is an mRNA modification catalyzed by the enzyme N-acetyltransferase 10 (NAT10), with position-dependent effects on mRNA translation. This protocol details a procedure to map ac4C at base resolution using NaBH4-induced reduction of ac4C and conversion to thymidine followed by sequencing (RedaC:T-seq). Total RNA is ribodepleted and then treated with NaBH4 to reduce ac4C to tetrahydro-ac4C, which specifically alters base pairing during cDNA synthesis, allowing the detection of ac4C at positions called as thymidine following Illumina sequencing. For complete details on the use and execution of this protocol, please refer to Arango et al. (2022).1

KW - Bioinformatics

KW - Molecular Biology

KW - RNAseq

KW - Sequencing

UR - http://www.scopus.com/inward/record.url?scp=85141964221&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85141964221&partnerID=8YFLogxK

U2 - 10.1016/j.xpro.2022.101858

DO - 10.1016/j.xpro.2022.101858

M3 - Article

C2 - 36595942

AN - SCOPUS:85141964221

SN - 2666-1667

VL - 3

JO - STAR Protocols

JF - STAR Protocols

IS - 4

M1 - 101858

ER -

Protocol for base resolution mapping of ac4C using RedaC:T-seq

Abstract

Funding

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this