ena-STUDY-UNIBO-27-11-2013-15:25:12:822-137
RNA-seq of 143B cybrids-derived xenografts
Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform. We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol. We tested the efficiency of our strategy by sequencing and analyzing the transcriptome of two well characterized xenograft tumor masses (OST), sharing the same nuclear genome, but carrying a different status of mitochondrial heteroplasmy and derived from the injection in nude mice of a human osteosarcoma cell line. The transcriptome sequencing of the two samples was performed by using two of the main sequencing platforms available on the market: 454 Roche and MiSeq Illumina.
Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform. We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol. We tested the efficiency of our strategy by sequencing and analyzing the transcriptome of two well characterized xenograft tumor masses (OST), sharing the same nuclear genome, but carrying a different status of mitochondrial heteroplasmy and derived from the injection in nude mice of a human osteosarcoma cell line. The transcriptome sequencing of the two samples was performed by using two of the main sequencing platforms available on the market: 454 Roche and MiSeq Illumina.