Strand-Specific RNA-Seq Libraries
RNA-Seq (conventional) after Poly-A enrichment or ribodepletion:
By default we generate strand-specific RNA-seq libraries. Strand-specific (also known as stranded or directional) RNA-seq libraries substantially enhance the value of an RNA-seq experiment. They add information on the originating strand and thus can precisely delineate the boundaries of transcripts in regions with genes on opposite strands.
There are several ways to accomplish strand-specificity. We incorporate dUTP during the second-strand synthesis of the cDNA. The dUTP containing strand will not be amplified by the proofreading polymerase used for library amplification, thus preserving the strand information for RNA-seq.
For single-end sequencing, the resulting data will represent the “anti-sense strand”. When using paired-end sequencing, the forward read of the resulting sequencing data represents the “anti-sense strand” and the reverse read the “sense strand” of the genes (for Trinity transcriptome assemblies the “–RF” orientation flag should be used). Illumina paired-end reads are always inward oriented (with the exception of “jumping” or “mate-pair” libraries).
Tag-Seq:
Tag-seq data are strand-specific and have a “sense-strand” orientation.
small-RNA-Seq/miRNA-seq:
Small RNA-seq data are strand-specific. The forward read of the sequencing data (read 1) is oriented as the reverse complement of the original RNA molecule. For libraries generated with the Revvity (PerkinElmer) Nextflex small RNA kits, the RNA sequence will be both preceded and followed by four bases with a random sequence.
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