How to Use T4 RNA Ligases with NEB E7330s for Small RNA Library Prep

How to Use T4 RNA Ligases with NEB E7330s for Small RNA Library Prep How to Use T4 RNA Ligases with NEB E7330s for Small RNA Library Prep

[NEB E7330s] Are the 3’ and 5’ Adaptors Pre-adenylated? What Ligase Should You Use?

When preparing small RNA libraries, choosing the correct adaptor format and ligase enzyme is critical for ligation efficiency and specificity. In this article, we’ll clarify the properties of the 3’ and 5’ adaptors in NEB’s E7330s kit and recommend the most suitable ligases for optimal performance.

✅ Are the 3’ and 5’ adaptors in the E7330s kit pre-adenylated?

  • 3’ adaptor: Pre-adenylated single-stranded DNA adaptor (5’rApp format)
  • 5’ adaptor: RNA adaptor, not pre-adenylated

These sequences are listed on page 16 of the official NEBNext manual.
📄 View NEB E7330s Manual

🔬 Why is pre-adenylation important?

Pre-adenylated adaptors are chemically modified to contain an adenyl group at their 5’ end (5’rApp structure). This allows ligation without ATP, which is critical when avoiding unwanted side reactions such as adaptor-dimer formation, concatemerization, or circle ligation. It ensures a cleaner ligation reaction, especially during 3' ligation of RNA.

🔧 Which ligase should you use?

Ligation efficiency depends on the directionality and chemical nature of your adaptor. Here’s the guideline:

Ligation Direction Adaptor Type Recommended Ligase
3’ ligation (ssRNA → pre-adenylated DNA adaptor) 3’ DNA adaptor T4 RNA Ligase 2, truncated K227Q (#M0351)
T4 RNA Ligase 2, truncated KQ (#M0373)
5’ ligation (RNA adaptor → ssRNA) 5’ RNA adaptor T4 RNA Ligase 1 (#M0204)

⚙️ Ligase Comparison Table

Ligase Mutation Use Case Advantages
M0242 (WT truncated) None Basic ligation with pre-adenylated adaptors Standard activity
M0351 (K227Q) K227Q Reduces unwanted concatemer formation Lower side-product formation
M0373 (KQ = R55K + K227Q) R55K, K227Q Highest ligation activity with specificity Best efficiency and minimal artifacts

💡 What's included in NEB #B0216?

  • T4 RNA Ligase Reaction Buffer
  • ATP (Adenosine 5’-Triphosphate)
  • 50% PEG 8000

Note: T4 RNA Ligase 2 does not require ATP during ligation with pre-adenylated adaptors.

🧪 Do I need to test both K227Q and KQ versions?

In most scenarios, testing both is unnecessary. NEB and many researchers now recommend T4 RNA Ligase 2, truncated KQ (M0373) due to its superior ligation performance and reduced formation of ligation artifacts. It’s ideal as a single-enzyme choice when working with pre-adenylated linkers.

🧭 Quick Troubleshooting Tips

  • Always use RNase-free conditions for ligation.
  • Denature RNA before adaptor ligation to avoid secondary structures.
  • Use PEG 8000 (final 10–15%) to enhance ligation efficiency.
  • For 3’ adaptor dimers, ensure adaptor concentration is optimized (not too high).

📌 Summary

Component Type Pre-adenylated? Recommended Ligase
3’ adaptor DNA ✅ Yes T4 RNA Ligase 2, truncated KQ
5’ adaptor RNA ❌ No T4 RNA Ligase 1

Accurate adaptor-ligase pairing is essential for high-quality small RNA library prep. Understanding enzyme mechanisms and adaptor chemistry will reduce failure rates and improve your downstream sequencing performance.

References: NEB E7330s Manual, RNA Ligase Selection Chart, NEB Product Pages

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