Stranded mRNA-Seq and ISO-Seq
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WHAT IS ISO-SEQ
ISO-Seq is a high-throughput sequencing technology that sequences full-length cDNA using Pac-Bio SMRT sequencing technology. ISO-Seq enables full length isoform RNA sequencing allowing for deeper characterization of full length transcripts. Briefly, RNA is transcribed into complementary DNA (cDNA) which serves as the base for SMRTbell library construction. Depending on the end goals of the sequencing effort additional rounds of amplification may be required before completing the rest of the library protocol. Samples are then sequenced on the Sequel II System. This method is a powerful tool for creating full length transcripts for downstream analysis for generation of full length transcriptomes to be used as genomic references for downstream experiments.
Common applications include:
Transcriptome characterization
De Novo whole transcriptome development
WHAT IS STRANDED RNA-SEQ?
Stranded RNA-seq is a high-throughput sequencing technology that retains information about the DNA strand from which a particular transcript RNA was derived. RNA is reverse transcribed into complementary DNA (cDNA) using dUPT second strand marking, which selectively amplifies the strand derived from the original transcript. This methodology is a powerful tool in understanding the genomic origins of RNA transcripts and is highly valuable for differential expression analysis.
Common applications include:
Differential expression analysis
Transcriptome assembly
EXAMPLE RNA-SEQ PROJECT
Differential expression analysis
Surveyed >70 fish samples of various organ tissue types, to understand population differences in gene expression
Used Nugen Universal Plus Prep kit
Sequenced on the Illumina Nova Seq 6000 with a 2x150bp SP chip
Looking for differential mRNA expression patterns across multiple populations