Which reads map to each strand in Stranded RNA workflows, and how is strandedness achieved?

In the Illumina Stranded mRNA, Illumina Stranded Total, TruSeq Stranded mRNA, and TruSeq Stranded Total workflows, Read 1 maps to the antisense strand and Read 2 maps to the sense strand.

• Note: In TruSeq Small RNA it is the opposite; Read1 maps to the sense strand and Read2 maps to the antisense strand.

  • TruSeq Small RNA achieves strandedness by ligating the RNA 5' Adapter to the 5' end of the transcript and the RNA 3' Adapter to the 3' end. After PCR addition of index adapters, this results in Read1 sequencing the sense orientation of the small RNA.

The following diagrams show how strandedness is achieved in the Illumina Stranded mRNA, Illumina Stranded Total, TruSeq Stranded mRNA, and TruSeq Stranded Total preparation kits. Note that the TruSeq RNA v2 workflow does not retain or provide stranded information.

Purify and Fragment mRNA or Deplete and Fragment Total RNA In Illumina/TruSeq Stranded mRNA workflows, oligo(dT) magnetic beads capture messenger RNAs (mRNAs) with polyA tails. In Illumina/TruSeq Stranded Total workflows, abundant RNA is depleted. Following mRNA purification or rRNA depletion, the RNA is fragmented into small pieces and primed with random hexamers.

Synthesize First Strand cDNA This step reverse transcribes the hexamer-primed RNA fragments to produce first strand complementary DNA (cDNA). The First Strand Synthesis Mix includes Actinomycin D, which allows RNA-dependent synthesis and improves strand specificity while preventing spurious DNA-dependent synthesis.

Synthesize Second Strand cDNA This step removes the RNA template and synthesizes a replacement strand to generate blunt-ended, double-stranded cDNA fragments. In place of deoxythymidine triphosphate (dTTP), deoxyuridine triphosphate (dUTP) is incorporated to quench the second strand during amplification and achieve strand specificity.

Adenylate 3' Ends This step adds an adenine (A) nucleotide to the 3สน ends of the blunt fragments to prevent them from ligating to each other during adapter ligation. A corresponding thymine (T) nucleotide on the 3สน end of the adapter provides a complementary overhang for ligating the adapter to the fragment.

Ligate Adapters or Anchors TruSeq Stranded mRNA and TruSeq Stranded Total: This process ligates multiple indexing adapters to the ends of the double-stranded cDNA fragments, which prepares them for hybridization onto a flow cell. Only the single-index adapter is shown in the following figure.

Illumina Stranded mRNA and Illumina Stranded Total: This step ligates pre-index anchors to the ends of the double-stranded cDNA fragments to prepare them for dual indexing. A subsequent PCR amplification step adds the index adapter sequences.

Amplify DNA Fragments TruSeq Stranded mRNA and TruSeq Stranded Total: The polymerase used in the assay does not incorporate past dUTP. Therefore, the second strand is effectively quenched during amplification. The products are enriched with PCR and purified to create the final cDNA library.

Illumina Stranded mRNA and Illumina Stranded Total: This step uses PCR to selectively amplify the anchor-ligated DNA fragments and add indexes and sequencing primer binding sequences for cluster generation. The polymerase used in the assay does not incorporate past dUTP. Therefore, the second strand is effectively quenched during amplification.

Final Library The library features single-index adapters, as shown in this workflow, or dual-index adapters, which contain an index at each end.

Cluster Generation and Read 1 Sequencing In Read 1, sequencing reads map to the antisense strand.

Paired-end Turnaround and Read 2 Sequencing In Read 2, sequencing reads map to the sense strand.

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