Thermolabile USER® II Enzyme Applications in Next Generation Sequencing Library Preparation (M5508)

Designed for use in library prep for DNA, ChIP DNA and RNA (but not Small RNA), the NEBNext Adaptors enable high-efficiency adaptor ligation and high library yields, with minimized adaptor-dimer formation. Incorporating a novel hairpin loop structure, the NEBNext Adaptor ligates with increased efficiency to end-repaired, dA-tailed DNA. The loop contains a dU, which is removed by treatment with USER Enzyme (or Thermolabile USER® II Enzyme), to open up the loop and make it available as a substrate for PCR.

If the NEBNext Adaptors are used, the loop must be cleaved in order to make the adaptor-ligated molecule available for PCR. This is accomplished by removal of the dU in the center of loop, using USER Enzyme or Thermolabile USER® II Enzyme.

NEBNext Ultra II DNA Library Prep Workflow
When the NEBNext Adaptor is used, a 15 minute incubation with USER Enzyme or Thermolabile USER® II Enzyme is required after ligation.

NEBNext RNA Library Prep Workflows
Directional:
The second strand is selectively removed by excision of the dUs in the second strand, using the USER Enzyme or Thermolabile USER® II Enzyme. At the same time, if the NEBNext Adaptors are used, the dU in the center of the NEBNext adaptor loop is removed, opening the molecule and making it available for PCR. The product of dU excision is a single stranded molecule, with different adaptor-derived sequences on each end.

Non-Directional:
If the NEBNext Adaptors are used, the loop must be cleaved in order to make the adaptor-ligated molecule available for PCR. This is accomplished by removal of the dU in the center of loop, using the USER Enzyme or Thermolabile USER® II Enzyme.