Feb 18, 2026
Version 2
Cut&Run library preparation V4 V.2
This protocol is a draft, published without a DOI.
- Joanna Fernandez1
- 1University of Sussex
Protocol Citation: Joanna Fernandez 2026. Cut&Run library preparation V4. protocols.io https://dx.doi.org/Version created by Joanna Fernandez
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 18, 2026
Last Modified: February 18, 2026
Protocol Integer ID: 243641
Keywords: nebnext ultraii dna library prep protocol for illumina, nebnext ultraii dna library prep protocol, nebnext multiplex oligos for illumina, nebnext multiplex oligo, library preparation v3 preface this protocol, expected protein, assay, soutoglou laboratory, library preparation v4 preface this protocol
Abstract
Preface
This protocol takes input material from the Cell Signalling CUT&RUN Assay Kit (86652S) and has been used in the Soutoglou Laboratory
This protocol is based very heavily on the protocol by Nan Liu (https://www.protocols.io/view/library-prep-for-cut-amp-run-with-nebnext-ultra-ii-bagaibse) which has been a vital source of information.
This protocol also lifts directly from the NEBNext UltraII DNA Library Prep Protocol for Illumina (NEB #E7645L), and the NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs Sets 1-5) (NEB #E6440S) user manuals.
Additional steps and adjusted incubation times are used in this protocol to optimise library yield and quality.
Before you begin:
1. Quantification of samples from the CUT&RUN assay kit is essential, a minimum of 5ng total is required.
2. qPCR to verify the assay has successfully enriched the expected protein is advisable prior to beginning this protocol.
Materials
REAGENTS
1. NEBNext‱ Ultra‱ II DNA Library Prep Kit for Illumina‱ (E7645L)
2. NEBNext‱ Multiplex Oligos for Illumina‱ (96 Unique Dual Index Primer Pairs) (E6440S)
3. NEB Luna‱ Universal qPCR Master Mix (M3003S)
4. NEBNext‱ Multiplex Oligos for Illumina‱ (Index Primers Set 1) (E7335S)
6. Agilent High Sensitivity D1000 Sample Buffer (5067-5603)
7. Beckman Coulter AMPure XP beads for DNA Cleanup (wsr-467777)
Quantification of Cut&RUN samples
Quantify cut&RUN samples using High Sensitivity D1000 ScreenTape(s) for Agilent TapeStation (#5067-5584, #5067-5603)
Total input volume of sample will be 25μl, therefore calculation input DNA concentration and decide accordingly the adaptor dilution to use.
In our experience, histone modification samples generally require 1:10 adaptor dilution, and other samples often require 1:25 adaptor dilution
Quantification on tape station can look like this:
This is a normal and expected range of concentrations in our experience. For example for samples A1-D1, 5μl of sample was used in the End Repair Reaction, and 25μl of sample for samples E1-H2 was used in the End Repair Reaction
End Repair Reeaction
Mix NEBNext End Prep reaction according to manufacturer’s protocol. Add the
following reagents to the sample:
| A | B | |
| COMPONENT | VOL | |
| (green) NEBNext Ultra II End Prep Enzyme Mix | 1.5ul | |
| (green) NEBNext Ultra II End Prep Reaction Buffer | 3.5ul | |
| Fragmented DNA | 25ul | |
| Total Volume | 30ul |
Set a 20μl pipette to 20μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
Note: It is important to mix well. The presence of a small amount of bubbles will not interfere with performance.
Place in a thermal cycler, with the heated lid set to ≥ 60°C, and run the following program:
30 minutes @ 20°C
60minutes @ 50°C Important to preserve smaller fragments therefore lower
Hold at 4°C
Adaptor Ligation
Prepare adaptor dilutions according to quantification in Step 1 and prepare master mix for adaptor ligation
Adaptor dilution from NEBNext protocol. This is critical for good quality NGS libraries
Add the following components directly to the End Prep Reaction Mixture.
| A | B | |
| COMPONENT | VOL 1x | |
| EndPrepReactionMixture | 30ul | |
| (red) NEBNext Adaptor for Illumina** | 1.25ul | |
| (red) NEBNext Ultra II Ligation Master Mix* | 15ul | |
| (red) NEBNext Ligation Enhancer | 0.5ul | |
| Total Volume | 46.75 | |
Set a 100μl or 200μl pipette to 40μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect all liquid from the sides of the tube.
(Caution: The NEBNext Ultra II Ligation Master Mix is very viscous. Care should be taken to ensure adequate mixing of the ligation reaction, as incomplete mixing will result in reduced ligation efficiency. The presence of a small amount of bubbles will not interfere with performance).
Incubate at 20°C for 15 minutes in a thermal cycler with the heated lid off (minimum 15 minutes, often we do 40 minutes)
Add 1.5 μl of • (red) USER‱ Enzyme to the ligation mixture
Mix well and incubate at 37°C for 15 minutes with the heated lid set to ≥ 47°C.
Cleanup of Adaptor-Ligated DNA
Bring AMPure XP beads to room temperature for 30 minutes before use and vortex well to mix.
Add 1.2X volume (60ul) of AMPureXP beads to the ligation reaction. Mix well by pipetting up and down at least 10 times. Incubate samples on bench top for at least 10 minutes at room temperature.
if ever you centrifuge, be very careful to stop before the beads settle.
Place the tube on PCR magnetic stand for 5 minutes/ until the beads are separate from the supernatant.
If handling > 8 samples, use multi channel for washes
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted DNA. (Caution: do not discard beads).
Add 200μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant.
Be careful not to disturb the beads that contain DNA targets. Beads can be left with 80% ethanol for minutes but do not leave them too long.
Repeat the previous step once. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
Air the dry beads for up to 5 minutes while the tube is on the magnetic stand with the lid open. Caution: Do not over-dry the beads.
I tend to find 3 minutes is enough if not plenty! Note that the concentration of DNA bound often influences how quickly the beads 'crack'
Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads into 19 μl of 10 mM Tris-HCl or 0.1X TE or ddH2O. Mix well on a vortex mixer or by pipetting up and down 10 times. Incubate for at least 10 minutes at room temperature.
Where concerned about yield, incubate longer. Since we will be trying a qPCR to determine number of cycles, prepare qPCR mix while this is incubating
Place the tube/plate on a magnetic stand. After 5 minutes (or when the solution is clear), transfer 15 μl to a new PCR tube for (amplification).
Take 2μl per sample to run qPCR, or else, this is a safe stop point. If pausing, DNA should be stored in fresh tube at -20°C
Cycle number determination qPCR
Using NEB Universal Primer and a barcode primer, set up Luna qPCR as follows. Each sample should be run once, and this can be used to check the number of PCR cycles necessary for final library PCR. This step is to avoid over amplification
| A | B | |
| 1x | ||
| Fp | 0.25 | |
| Rp | 0.25 | |
| luna | 5 | |
| template | 2 | |
| H2o | 2.5 | |
| Total | 10 |
NEB LUNA qPCR mix
| A | B | C | D | |
| CYCLE STEP | TEMP | TIME | CYCLES | |
| Initial Denaturation | 95°C | 60 seconds | 1 | |
| Denaturation | 95°C | 15 seconds | ||
| Extension | 60°C | 30 | 40-45 | |
| Melt curve | 60°C-95°C | various | 1 |
Analyse qPCR and consider Cqs for each sample. The Cq +2 is usually an appropriate number of PCR cycles for barcode PCR step
PCR Enrichment of Adaptor-ligated DNA
Before beginning, prepare index primers:
Remove 96 Unique Dual Index Primers Plate from freezer and thaw for 10-15 minutes at room temperature
Remove hard plastic cover. If it is a new plate, vortex plate and centrifuge (280 x g for approximately 1 minute). Else, just centrifuge.
Orient the plate with red line facing user, and label sample numbers. Make sure to take note of which well was used for which primer.
Prior to pipetting the primer mix, use a new, clean pipette tip to pierce the foil. Then use a fresh pipette tip to remove the necessary volume.
Prepare PCR mix by adding the following components to a sterile strip tube:
| A | B | |
| COMPONENT | VOL | |
| (blue) Dual Index Primer Mix* | 10ul | |
| (blue) NEBNext Ultra II Q5 Master Mix | 25ul | |
| DNA from previous | 15ul | |
| Total Volume | 50ul |
Mix the reactions thoroughly and perform a quick spin to collect all liquid from the sides of the tube
Place the reactions in a thermal cycler and perform the PCR amplification using the following PCR conditions. Set the lid to at least 98°C
Thermal cycler conditions from NEBNEXT ULTRAII
PCR Clean up
Allow AMPureXP beads to equilibrate to room temperature for 30 minutes and vortex to resuspend.
Add 45 μl (0.9X) resuspended beads to the PCR reaction. Mix well by pipetting up and down at least 10 times. Be careful to expel all of the liquid out of the tip during the last mix. Vortexing for 3-5 seconds on high can also be used. If centrifuging samples after mixing, be sure to stop the centrifugation before the beads start to settle out.
Incubate samples on bench top for at least 10 minutes at room temperature.
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing on the magnetic stand.
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution: do not discard the beads).
Add 200 μl of 80% freshly prepared ethanol to the tube/plate while in the magnetic stand. Incubate at room temperature for 30 seconds, and then carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
Repeat Step 31. once for a total of two washes. Be sure to remove all visible liquid after the second wash. If necessary, briefly spin the tube/plate, place back on the magnet and remove traces of ethanol with a p10 pipette tip.
Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand
with the lid open.
Caution: Do not over-dry the beads. This may result in lower recovery of DNA. Elute the samples when the beads are still dark brown and glossy looking, but when all visible liquid has evaporated. When the beads turn lighter brown and start to crack, they are too dry.
Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 μl of 1X TE or ddH2O.
Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 10 minutes at room temperature. If necessary, quickly spin the sample to collect the liquid from the sides of the tube or plate wells before placing back on the magnetic stand.
Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer μl to a new PCR tube and store at –20°C.
Quantification
Check the size distribution with Agilent TapeStation HS dsDNA 1000 (#5067-5584, #5067-5603). The sample may need to be diluted minimum 1:10 before loading. Aim for quantification of >2nM within region table per sample.
Illumina NEXTSeq1000
Libraries should be diluted and pooled according to guidelines from Illumina.
Analysis pipeline
Basic data analysis is conducted as outlined in https://github.com/joannafernandez/cut-run_for_soutoglou_laboratory
Protocol references
1. Liu et al. (2021). Library Prep for CUT&RUN with NEBNext‱ Ultra‱ II DNA Library Prep Kit for Illumina‱ (E7645) V.2 . protocols.io DOI: https://dx.doi.org/10.17504/protocols.io.bagaibse
2. New England Biolabs. (2022). NEBNext‱ Ultra‱ II DNA Library Prep Kit for Illumina‱version 7.0_9/22 – Instruction Manual. New England Biolabs. Retrieved from https://www.neb.com/E7645
3. New England Biolabs. (2024). NEBNext‱ Multiplex Oligos for Illumina‱(96 Unique Dual Index Primer Pairs Sets 1–5) NEB #E6440S/L, NEB #E6442S/L, NEB #E6444S/L, NEB #E6446S/L, NEB #E6448S/L version 10.0_5/24 – Instruction Manual. New England Biolabs. Retrieved from https://www.neb.com/en-gb/products/e6440
4. New England Biolabs. (2020) Luna‱ Universal qPCR Master Mix NEB #M3003S/L/X/E version 3.0_3/20 – Instruction Manual. New England Biolabs. Retrieved from https://www.neb.com/en-gb/products/m3003-luna-universal-qpcr-master-mix