Library Prep Partners
      Back to home
      1. Singular Knowledge Base
      2. Library Preparation
      3. Library Prep Partners

      NEBNext Ultra II Directional RNA Library Prep Kit

      Q: How do I adapt the NEBNext Ultra II Directional RNA Library Prep Kit and the NEBNext rRNA Depletion Kit v2 (Human/ Mouse/ Rat) to sequence on the G4 Sequencing Platform?

      A: Follow the NEBNext Ultra II Directional RNA Library Prep protocol for use with the NEBNext rRNA Depletion Kit v2 (Human/ Mouse/ Rat) from New England Biolabs (NEB), with the following modifications:

      LIBRARY PREP PROTOCOL

      1. Perform section 2 of the NEBNext Ultra II Directional RNA Library Prep Protocol with the rRNA Depletion Kit v2 until you reach the Adaptor Ligation step.

      NOTE: Use the Singular Genomics (SG) Universal Library Prep Adapter in the Adaptor Ligation step listed in the protocol as follows:

      1. Dilute the SG Adapter depending on the amount of total input RNA used:
      Reagent
      5 ng–9.9 ng input RNA
      10 ng–100 ng input RNA
      101 ng–1000 ng input RNA
      SG Universal Library Prep Adapter (15 µM)
      1 µL
      1 µL
      1 µL
      Nuclease-free water
      199 µL
      24 µL
      4 µL
      Total
      200 µL
      25 µL
      5 µL
      Dilution
      200-fold
      25-fold
      5-fold
      1. Add SG Adapter, NEBNext Ultra II FS Ligation Enhancer, and NEBNext Ultra II Ligation Master Mix to the tube with fragmented and end-prepped DNA:
      Reagent
      5 ng–9.9 ng input RNA
      10 ng–100 ng input RNA
      101 ng–1000 ng input RNA
      Fragmented and end prepped DNA
      60 µL
      60 µL
      60 µL
      SG Universal Library Prep Adapter
      2.5 µL (200-fold dilution)
      2.5 µL (25-fold dilution)
      2.5 µL (5-fold dilution)
      NEBNext Ligation Enhancer
      1 µL
      1 µL
      1 µL
      NEBNext Ultra II Ligation Master Mix
      30 µL
      30 µL
      30 µL
      Volume
      93.5 µL
      93.5 µL
      93.5 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 Enzyme to the ligation mixture.
      1. Pipette up and down 10 times to mix thoroughly and spin down briefly.
      2. Incubate at 37°C for 15 minutes with the heated lid set to at least 45°C.
      3. Immediately proceed with the Purification of the Ligation Reaction step in the NEBNext protocol.

      NOTE: Perform PCR amplification with 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 PCR Primers (5 µM each)
      10 µL
      Total
      50 µL
      1. Perform the library Quality Control steps listed here: Library Prep Quality Control

      DEMONSTRATED G4 SEQUENCING EXPERIMENTS

      For a demonstrated NEBNext Ultra II Directional RNA sequencing experiments on G4, see the App Note RNA Sequencing Using the New England Biolabs (NEB) Ultra ll RNA Library Prep Kit and G4 Sequencing Platform .

      ORDERING INFORMATION

      Singular Genomics (SG) indexed and non-indexed PCR primers and adapters can be purchased 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 Directional RNA Library Prep Kit can be purchased from NEB:

      Part #
      Product
      NEBNext Ultra II Directional RNA Library Prep Kit for Illumina
      NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with Sample Purification Beads

      RNA Depletion Kit v2 (Human/ Mouse/ Rat) can be purchased from NEB:

      Part #
      Product
      RNA Depletion Kit v2 (Human/ Mouse/ Rat)
      RNA Depletion Kit v2 (Human/ Mouse/ Rat) with Sample Purification Beads

      Version: 29 Sep 2023