| Abstract |
Sequencing-based analysis of 5â²Â capped ends of RNAs have unveiled an unexpected complexity of the mammalian transcriptome, in which coding and non-coding RNAs produced in loci often harboring multiple promoters coexist with processed RNAs that exhibit a variety of 5â² modifications. In light of .. [more]these discoveries, assessing whether novel transcription start sites (TSSs) are alternative promoters of known genes or promoters of newly identified transcripts require dedicated approaches. Furthermore, collecting such data from limiting amount of RNAs, such as microdissected cell types or from sub-cellular compartments with ex vivo samplesâa necessary step to reduce the complexity of the studied transcriptome while keeping its biological relevanceâoften requires quantities of starting total RNA that are not sufficient for current protocols. Addressing both challenges, we developed nanoCAGE, a 5â²Â ends RNA profiling method of both poly-A+ and poly-Aâ transcripts that operates using as little as 10 ng of total RNA, coupled with CAGEscan, a technology for a gene-agnostic exploration of the transcriptome structure and dynamics, linking de novo-assembled transcripts to their regulatory regions [less]
|
| Description |
While the classic CAGE protocol requires a substantial number of biochemical processing steps, CAGEscan and nanoCAGE take advantage of a peculiar property of reverse transcriptase, called âtemplate switchingâ to select 5â²Â ends of capped transcripts. This property allows proceeding directly fr .. [more]om the first-strand cDNA synthesis to the addition of the second-strand cDNA primer at the 5â²Â end of the original RNA sequence without purification steps, thereby avoiding loss of material. This approach exploits the ability of the reverse-transcriptase to extend the cDNA using the mRNAâs cap as a template: the resulting neo-synthesized first strand cDNA carries one to three C nucleotides in correspondence of the cap structure. These Cs are hybridized to the ribo-G at the 3â²Â end of a template switching (TS) oligonucleotide. Then, the reverse transcriptase extends cDNA polymerization using the TS oligonucleotide as template, providing extra 3â² sequences to the first-strand cDNA. Due to the selectivity of the template switch for capped molecules, both protocols were used on total RNA, without depletion of ribosomal RNAs. Notably, the usage of 3â²Â random primer allows the detection of non-coding, non-polyadenylated RNAs. In the CAGEscan protocol, cDNAs are not cleaved, but directly prepared for sequencing by a second PCR which adds the adapter sequences needed to bridge PCR in the Illumina Genome Analyzers. In contrast with the nanoCAGE protocol, sequencing of both the 5â²Â end and the 3â²Â end of the template-switched captured cDNAs yields collections of 3â²Â ends reads âscanningâ transcripts that can be unequivocally defined by their common 5â²Â end. Yet, unlike nanoCAGE libraries that contain uniformly short sequences, CAGEscan libraries are composed of sequences of broader size range including fragments longer than 1 kb, which poorly perform on currently available second-generation sequencing platform. However, since CAGEscan exclusively uses random primers at high concentration and commercial reverse transcriptases do not have a strong strand displacement activity, CAGEscan sequencing templates prepared by our optimized protocol are kept relatively short, regardless the length of the original mRNA molecules. [less]
|
FTP
