Home FAQs Can I use the NEBNext Low-bias Small RNA Library Prep Kit to make Ribo-seq libraries?

FAQ: Can I use the NEBNext Low-bias Small RNA Library Prep Kit to make Ribo-seq libraries?

Yes, the NEBNext Low-bias Small RNA Library Prep Kit can be used to prepare Ribo-seq libraries. Please review the following general guidance for both the generation of ribosome protected fragments (RPFs) and library preparation, based on customer feedback:

  1. When choosing the RNase or nuclease for the ribosome footprinting, consider trying an RNase that does not have a strong bias to certain nucleotides at the cleavage site. Since this kit uses randomized splint adaptors, if there is a strong bias for a specific base as either the 5' or 3' end of the RNA, capture may not be as efficient. The pool of adaptors that complement the specific base bias may be quickly depleted, and the noncomplementary excess adaptors may lead to increased adaptor-dimer formation.

    2.  

    ✅ RNase I is a good example of a choice with less sequence bias.

    ❌ RNase T1 is an example of a choice with a strong sequence bias.

     

  2. Many common RNases leave ends that are not compatible with our library chemistry and, therefore, RNA will need to be end repaired to have 5' monophosphate and 3' hydroxyl ends prior to starting the NEBNext Low-bias Small RNA workflow. T4 PNK can be used for end repair. First, use the phosphatase activity of PNK in the absence of ATP to generate 3'OH ends. Then, a subsequent PNK reaction that includes ATP, will produce the 5’ phosphate. Contact NEB technical support for a comprehensive protocol.

  3. The end-repaired, ribosome-protected fragments should follow the recommendations for enriched small RNA samples for adaptor dilutions and PCR cycling throughout the manual.

  4. For purified ribosome-protected fragments, you can use either one of the following modifications to the Bead Size Selection after the Reverse Transcription Reaction (Section 6). If you are starting with very low input and are concerned about final library yields, Option 2 is recommended.

    5. Option 1: Less-stringent size selection

    1. At step 6.2 add 37 µL of NEBNext Sample Purification Beads.
    2. At step 6.7 add 69 µL of NEBNext Sample Purification Beads.

        ** Other than these two bead volume modifications, follow the rest of Section 6 as written.

    Option 2: Bead cleanup

    1. Start at step 6.7 and add 106 µL (2X) of NEBNext Sample Purification Beads to the Reverse Transcription reaction.

        ** Continue through the remaining steps of this section as written.


  5. Follow Section 8B for the bead cleanup of the final libraries.

If you have any questions about our this guidance, or you need additional support, please contact our dedicated Technical Support team: https://www.neb.com/en-us/forms/neb-technical-support.