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      NEBNext Ultra II FS DNA Library Prep Kit

      Q: How do I adapt the NEBNext Ultra II FS DNA Library Prep Kit to sequence on the G4 Sequencing Platform?

      A: Follow the NEBNext Ultra II FS DNA library prep protocol from New England Biolabs (NEB) with the following modifications:

      LIBRARY PREP PROTOCOL

      1. Perform one of the following NEBNext protocols until you reach the Adaptor Ligation step.

      NOTE: Instead of the adapter used in the Adaptor Ligation step listed in the protocol, use the Singular Genomics (SG) Universal Library Prep Adapter as listed below.

      Adapter Dilutions: For input DNA < 100 ng, dilute the adapter to 1.5 µM or 0.6 µM with nuclease-free water, depending on the amount of input DNA at the fragmentation step:
      Less than 5 ng input DNA
      5–99.9 ng input DNA
      100–500 ng input DNA
      SG Universal Library Prep Adapter (15 µM)
      1 µL
      1 µL
      Use adapter undiluted
      Nuclease-free water
      24 µL
      9 µL

      Total
      25 µL
      10 µL

      Final adapter concentration
      0.6 µM
      1.5 µM

      1. Pipette the NEBNext Ultra II FS Ligation Master Mix up and down 10 times before use to mix. The presence of a small number of bubbles will not interfere with performance.
      2. Add adapter, NEBNext Ultra II FS Ligation Enhancer, and NEBNext Ultra II Ligation Master Mix to the tube with DNA:
      Reagent
      Less than 5 ng input DNA
      5–99.9 ng input DNA
      100–500 ng input DNA
      Fragmented and end-prepped DNA
      35 µL
      35 µL
      35 µL
      NEBNext Ultra II Ligation Master Mix
      30 µL
      30 µL
      30 µL
      NEBNext Ligation Enhancer
      1 µL
      1 µL
      1 µL
      SG Universal Library Prep Adapter
      2.5 µL (0.6 µM dilution)
      2.5 µL (1.5 µM dilution)
      2.5 µL (undiluted)
      Total
      68.5 µL
      68.5 µL
      68.5 µL
      1. Pipette up and down 10 times to mix thoroughly and spin down briefly.
      2. Incubate for 15 minutes at 20° C on a covered block or thermocycler with the heated lid off.
      3. Add 3 µl of USER to the ligation mixture.
      4. Pipette up and down 10 times to mix thoroughly and spin down briefly.
      5. Mix well and incubate at 37°C for 15 minutes with the heated lid set to at least 47°C.
      6. After the samples have cooled, proceed with the size selection/cleanup step in the NEBNext protocol.

      NOTE: Perform PCR amplification with indexed or non-indexed SG primers in the PCR Enrichment of Adapter-ligated DNA step instead of the primers listed in the protocol.

      1. Use the following PCR reagents for the PCR Enrichment of Adapter-ligated DNA step:
      Reagent
      Volume
      Cleaned up DNA
      15 µL
      Q5 Master Mix
      25 µL
      SG UDI Primers (5 µM each)
      10 µL
      Total
      50 µL
      1. Perform the library Quality Control steps listed here: Amplified Library Prep Quality Control

      DEMONSTRATED G4 SEQUENCING EXPERIMENTS

      For demonstrated NEBNext Ultra II FS DNA Library Prep Kit sequencing experiments on G4, see the App Note

      DNA Sequencing on the G4 Sequencing Platform Using the NEBNext Ultra II FS DNA Library Prep Kit .

      ORDERING INFORMATION

      Indexed or non-indexed SG PCR primers and SG Universal Library Prep Adapter from Singular Genomics:

      Part #
      Product
      SG Universal Library Prep Adapter + UDI Primers [1–96]
      SG Universal Library Prep Adapter + UDI Primers Set A [1–24]
      SG Universal Library Prep Adapter + UDI Primers Set B [25–48]
      SG Universal Library Prep Adapter (24 Rxns)
      SG Non-Indexed Library Prep Primers (24 Rxns)
      SG Universal Library Prep Adapter (96 Rxns)
      SG Non-Indexed Library Prep Primers (24 Rxns)

      NEBNext Ultra II FS DNA Library Prep Kit from NEB:

      Part #
      Product
      NEBNext Ultra II FS DNA Library Prep Kit for Illumina
      NEBNext Ultra II FS DNA Library Prep Kit for Illumina with Sample Purification Beads

      USER II Enzyme from NEB:

      Part #
      Product
      Thermolabile USER II Enzyme

      Version: 19 Mar 2024