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.

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