A multiplex RNA-seq strategy to profile poly(A+) RNA: application to analysis of transcription response and 3' end formation.
|Title||A multiplex RNA-seq strategy to profile poly(A+) RNA: application to analysis of transcription response and 3' end formation.|
|Publication Type||Journal Article|
|Year of Publication||2011|
|Authors||Fox-Walsh K, Davis-Turak J, Zhou Y, Li H, Fu X-D|
|Date Published||2011 Oct|
|Keywords||3' Untranslated Regions, Gene Expression Profiling, HeLa Cells, High-Throughput Nucleotide Sequencing, Humans, Oligonucleotide Array Sequence Analysis, Poly A, Polyadenylation, RNA, RNA-Binding Protein FUS, Sequence Analysis, RNA, Transcriptome|
RNA-seq technologies are now replacing microarrays for profiling gene expression. Here we describe a robust RNA-seq strategy for multiplex analysis of RNA samples based on deep sequencing. First, an oligo-dT linked to an adaptor sequence is used to prime cDNA synthesis. Upon solid phase selection, second strand synthesis is initiated using a random primer linked to another adaptor sequence. Finally, the library is released from the beads and amplified using a bar-coded primer together with a common primer. This method, referred to as Multiplex Analysis of PolyA-linked Sequences (MAPS), preserves strand information, permits rapid identification of potentially new polyadenylation sites, and profiles gene expression in a highly cost effective manner. We have applied this technology to determine the transcriptome response to knockdown of the RNA binding protein TLS, and compared the result to current microarray technology, demonstrating the ability of MAPS to robustly detect regulated gene expression.