TruSeq Stranded RNA Kit FAQs: differences from TruSeq RNA Library Prep Kit v2

Why is there no longer an end repair step? End repair now happens during second strand cDNA synthesis, thereby eliminating the need for a separate end repair step and shortening the protocol.

Are dual index RNA adapter plates compatible with the TruSeq RNA Library Prep kit v2? The TruSeq RNA dual index plates have not been officially validated with the TruSeq RNA v2 library prep kit, though are generally interchangeable with the TruSeq Single Index RNA adapters. Users may need to optimize the final bead clean up step by slightly reducing the final bead volume used if transitioning to dual indexed adapters.

Why are library yields lower when using the TruSeq Stranded mRNA HT kit and protocol as compared to TruSeq RNA Library Prep Kit v2 with the same input? Users performing sample prep with the TruSeq Stranded mRNA HT kit with 100 ng input of total RNA may experience lower library yields as compared to TruSeq v2 with the same input RNA. There are two possible reasons for this: First, the TruSeq Stranded mRNA Sample Prep offers strand information on the mRNA, to accomplish this, only the sequence that is transcribed is converted into library as compared to the TruSeq v2 sample prep where both the transcribed sequence and its reverse complement are converted into library. For this reason, half as much of the mRNA is converted into library so the library yields are generally lower. Second, the dual-index HT adapters are longer than the v2/LT adapters so their performance in the ligation step may be different.

For these two reasons, users may find that their library yields with the TruSeq Stranded mRNA HT kit are ~50% of the TruSeq v2 kit. Even at the reduced yield, there is still enough library to quantify and sequence, so it is more of a cosmetic problem. One mitigation is to perform the HS (high sample) method (midi plate/shaking) vs. the LS (low sample) method (PCR plate, pipette mixing) method. Generally, yields are higher with the HS method, and we recommend in the User Guide that if >24 samples preps are being performed with the HT kit, then the HS method should be used. Generally, this is not a concern for the TruSeq Stranded Total RNA HT kit since overall yields are 2-4x with that kit compared to the mRNA kit.

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