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Development of Poly(A)-ClickSeq as a Tool Enabling Simultaneous Genome-wide Poly(A)-site identification and Differential Expression Analysis
- Source :
- Methods
- Publication Year :
- 2019
-
Abstract
- The use of RNA-seq as a generalized tool to measure the differential expression of genes has essentially replaced the use of the microarray. Despite the acknowledged technical advantages to this approach, RNA-seq library preparation remains mostly conducted by core facilities rather than in the laboratory due to the infrastructure, expertise and time required per sample. We have recently described two ‘click-chemistry’ based library construction methods termed ClickSeq and poly(A)-click-seq (PAC-seq) as alternatives to conventional RNA-seq that are both cost effective and rely on straightforward reagents readily available to most labs. ClickSeq is random-primed and can sequence any (unfragmented) RNA template, while PAC-seq is targeted to poly(A) tails of mRNAs. Here, we further develop PAC-seq as a platform that allows for simultaneous mapping of poly(A) sites and the measurement of differential expression of genes. We provide a detailed protocol, descriptions of appropriate computational pipelines, and a proof-of-principle dataset to illustrate the technique. PAC-seq offers a unique advantage over other 3’ end mapping protocols in that it does not require additional purification, selection, or fragmentation steps allowing sample preparation directly from crude total cellular RNA. We have shown that PAC-seq is able to accurately and sensitively count transcripts for differential gene expression analysis, as well as identify alternative poly(A) sites and determine the precise nucleotides of the poly(A) tail boundaries.
- Subjects :
- Polyadenylation
Computer science
genetic processes
Genome, Insect
Computational biology
Genome
General Biochemistry, Genetics and Molecular Biology
Article
Market fragmentation
03 medical and health sciences
Gene expression
Animals
natural sciences
3' Flanking Region
RNA, Messenger
Molecular Biology
Gene
Cells, Cultured
030304 developmental biology
Gene Library
Protocol (science)
0303 health sciences
Sequence Analysis, RNA
Gene Expression Profiling
030302 biochemistry & molecular biology
RNA
TheoryofComputation_GENERAL
High-Throughput Nucleotide Sequencing
Identification (information)
TheoryofComputation_MATHEMATICALLOGICANDFORMALLANGUAGES
ComputingMethodologies_PATTERNRECOGNITION
Drosophila melanogaster
Gene Expression Regulation
Insect Proteins
Click Chemistry
Poly A
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Methods
- Accession number :
- edsair.doi.dedup.....589f249475b444833d067f5e859601be