TITLE: Library Prep for CUT&RUN with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645)
AUTHORS: Nan Liu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = ":UNDERLINE;">Choose your protocol:</span></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/cwgqjgf6.png" /></div><div class = "text-block">For Henikoff lab protocol, please see:</div><div class = "text-block"><span style = ":UNDERLINE;">Why?</span></div><div class = "text-block">The difference between NEB stem loop adaptors and illumina adaptors are their lengths. When ligated to both ends of DNA, they add 64bp and 124bp respectively. This makes huge difference when you do beads based cleaning up.</div><div class = "text-block"><span style = ":UNDERLINE;">What is this protocol?</span></div><div class = "text-block"><span>This protocol is based on library preparation manual of “NEBNext® Ultra™ II DNA Library Prep Kit for Illumina”, NEB E7645, and is specifically modified to make libraries from small DNA fragments (25-150bp). We have successfully applied this protocol to make libraries for </span><a href="https://www.protocols.io/view/cut-run-targeted-in-situ-genome-wide-profiling-wit-mgjc3un" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">CUT&RUN</span></a><span> experiments and showed that it greatly increased the preservation of small fragments (Liu, Cell 2018. Fig. S2B [</span><a href="http://the_brain.bwh.harvard.edu/pubs/Liu_Cell2018_BCL11A_Epub.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">pdf</span></a><span>])</span></div></div>

[STEPS]
?. [NEBNext End Prep]
Important Note: Please use manufacturer protocol if you are making histone modification CUT&RUN library. Do not use this one, which is optimized for small fragments.Mix componentsAdd the following components to a sterile nuclease-free tube:  AB1ReagentVolume2CUT&RUN DNA (6ng)25 μl3(green) NEBNext Ultra II End Prep Enzyme Mix1.5 μl4(green) NEBNext Ultra II End Prep Reaction Buffer3.5 μl
AB1ReagentVolume2CUT&RUN DNA (6ng)25 μl3(green) NEBNext Ultra II End Prep Enzyme Mix1.5 μl4(green) NEBNext Ultra II End Prep Reaction Buffer3.5 μl
Starting Material
Set a 100 or 200 μl pipette to 25 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect the liquid from the side of the tube. Note: it is important to mix well. The presence of a small amount of bubbles will not interfere with the performance.
?. [NEBNext End Prep]
Place in a thermocycler, with the heated lid set to ≥ 60°C, and run the following program:  AB1TimeTemperature230 minutes20°C360 minutes50°C4Hold4°C
AB1TimeTemperature230 minutes20°C360 minutes50°C4Hold4°C
?. [Adaptor Ligation]
Dilute the Adaptor based on the amount you need to use. I dilute the original 15 μM adaptor to 3 μM when I work with 6 ng input DNA.
If your input is 6 ng, its molarity is 0.2 pmol. I typically use ~4 pmol (3 μM x 1.25 μL) of Adaptor, which is 20 fold of DNA fragment. Lower amount of Adaptor can reduce the amount of final PCR dimers. If you have severe dimer contamination, you can use as low as 0.6 pmol Adaptor for 0.2 pmol of input DNA (3x).
?. [Adaptor Ligation]
Add the following components directly to the End Prep Reaction Mixture:  AB1ReagentVolume2(red) NEBNext Ultra II Ligation Master Mix15&nbsp;μl3(red) NEBNext Ligation Enhancer0.5 μl4(red) NEBNext Adaptor For Illumina1.25 μl
AB1ReagentVolume2(red) NEBNext Ultra II Ligation Master Mix15&nbsp;μl3(red) NEBNext Ligation Enhancer0.5 μl4(red) NEBNext Adaptor For Illumina1.25 μl
The NEBNext adaptor is provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335,  #E7500,  #E7710, #E7730, #E6609, #E7600) Oligos for Illumina. There is only one universal Adaptor, and the barcodes are embedded in the PCR primers. The Ligation Master Mix and Ligation Enhancer can be mixed ahead of time and is stable for at least 8 hours @ 4°C. We do not recommend adding adaptor to a premix in the Adaptor Ligation Step. For best results add adaptor last and immediately mix well, or premix adaptor and sample and then add the other ligation reagents.
Set a 100 or 200 μl pipette to 40 μl and then pipette the entire volume up and down at least 10 times to mix thoroughly. Perform a quick spin to collect the liquid from the side of the tube. Note: it is important to mix well. The presence of a small amount of bubbles will not interfere with the performance.
?. [Adaptor Ligation]
Incubate at 20°C for 15 minutes in a thermal cycler.
?. [Adaptor Ligation]
Add 1.5 μl of USER™ enzyme to the ligation mixture. Mix well and incubate at 37°C for 15 minutes with heated lid set to ≥ 47°C.
USER addition and USER incubation are only required for use with NEBNext adaptors. USER is provided in the NEBNext Index Primer kits. It is also available as a separate product NEB# M5505 which can be used in NEBNext library prep workflows.
?. [Cleanup of Adaptor-ligated DNA]
Vortex AMPure XP beads to resuspend. Please allow the beads to warm to room temperature for at least 30 minutes before use.
We haven't tested SPRIselect beads. Only substitute if you are sure they work exactly the same.
?. [Cleanup of Adaptor-ligated DNA]
Add 80 μl (~1.75x) AMPure XP beads to ligation reaction. Mix well by pipetting up and down at least 10 times. Incubate samples on bench top for at least 5 minutes at room temperature.
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.CRITICAL: Use of 80 μl AMPure XP beads is essential to purify short DNA.
?. [Cleanup of Adaptor-ligated DNA]
Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
?. [Cleanup of Adaptor-ligated DNA]
After 5 minutes (or when the solution is clear), carefully remove and discard the supernatant that contains unwanted DNA. (Caution: do not discard beads).
Be careful not to disturb the beads that contain the desired DNA targets
?. [Cleanup of Adaptor-ligated DNA]
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. Beads can be left with 80% ethanol for minutes but we do not recommend to leave them too long.
?. [Cleanup of Adaptor-ligated DNA]
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.
?. [Cleanup of Adaptor-ligated DNA]
Air the dry beads for up to 5 minutes while the tube is on the magnetic stand with the lid open. Caution: Do not overdry the beads.
?. [Cleanup of Adaptor-ligated DNA]
Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads into 15 μl of 10 mM Tris-HCl or 0.1X TE. Mix well on a vortex mixer or by pipetting up and down 10 times. Incubate for at least 2 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.
?. [Cleanup of Adaptor-ligated DNA]
Place the tube/plate on a magnetic stand. After 5 minutes (or when the solution is clear), transfer 13 μl to a new PCR tube for (amplification).
SAFE STOP: Samples can be stored at -20ºC
?. [PCR Amplification]
Mix the following components in sterile strip tubes (Total volume  30 μl):  AB1ReagentVolume2Adaptor Ligated Fragments13 µl3(blue) NEBNext Ultra II Q5 Master Mix15 µl4(blue) Index Primer/i7 Primer1 µl5(blue) Universal PCR Primer/i5 Primer1 µl
AB1ReagentVolume2Adaptor Ligated Fragments13 µl3(blue) NEBNext Ultra II Q5 Master Mix15 µl4(blue) Index Primer/i7 Primer1 µl5(blue) Universal PCR Primer/i5 Primer1 µl
The primers are provided in NEBNext Singleplex (NEB #E7350) or Multiplex (NEB #E7335, #E7500, #E7710, #E7730, #E6609, #E7600) Oligos for Illumina.
?. [PCR Amplification]
PCR using the following cycling conditions:  ABCD1Cycle StepTemperatureTimeCycles2Initial Denaturation98°C30 Seconds13Denaturation98°C10 Seconds12*4Annealing/ Extension65°C10 Seconds5Final Extension65°C5 Minutes16Hold4°C∞&nbsp;
ABCD1Cycle StepTemperatureTimeCycles2Initial Denaturation98°C30 Seconds13Denaturation98°C10 Seconds12*4Annealing/ Extension65°C10 Seconds5Final Extension65°C5 Minutes16Hold4°C∞&nbsp;
When starting with 6 ng DNA, I’ve obtained 10-40 ng of DNA with 12 cycles of amplification with different samples.If you use less input, consider increasing the cycle number.
?. [Cleanup of PCR Amplification]
Vortex AMPure XP beads to resuspend. Please allow the beads to warm to room temperature for at least 30 minutes before use.
We haven't tested SPRIselect beads. Only substitute if you are sure they work exactly the same.Important Note: Please use manufacturer protocol if you are making histone modification CUT&RUN library. The following size selection method is optimized for small fragments.
?. [Cleanup of PCR Amplification]
First round size selection: remove PCR products that are >350 bp. Add 24 μl (0.8X) resuspended AMPure XP beads to the PCR reaction. Mix well by pipetting up and down at least 10 times.Incubate for at least 5 minutes at room temperature.
PCR products larger than 350 bp, which correspond to fragment size of 220 bp, will be removed during this step. If you want to preserve these information, use less AMPure beads or skip first round size selection. You will need to adjust the amount of AMPure beads accordingly during the second round size selection. Final AMPure is 1.2x volume of the PCR product.CRITICAL: such adjustment requires a good understanding of the way AMPure beads works. See note in step 22 for more information.
?. [Cleanup of PCR Amplification]
Place the tube/plate on an appropriate magnetic stand to separate the beads from the supernatant (5 min or when the solution is clear).
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.
?. [Cleanup of PCR Amplification]
After 5 minutes (or when the solution is clear), carefully transfer the supernatant containing your DNA to a new tube (Caution: do not discard the supernatant). Discard the beads that contain the unwanted large fragments.
?. [Cleanup of PCR Amplification]
Second round of size selection: remove PCR products shorter than 150 bp.Add 12 μl (final 1.2x) resuspended AMPure XP beads to the supernatant and mix at least 10 times. Then incubate samples on the bench top for at least 5 minutes at room temperature.
Be careful to expel all of the liquid from the tip during the last mix.This post is very helpful for understanding how AMPure beads size selection work. The ratio here, 1.2x, is the final ratio of PEG/NaCl solution to PCR product. The 24 μL PEG/NaCl solution (supernatant from step 21) is combined with 12 μL new beads, adding up to 36 μL PEG/NaCl. This is 1.2x to 30 μL PCR product. The ratio determines the size of DNA to be captured by the beads.
?. [Cleanup of PCR Amplification]
Place the tube/plate on an appropriate magnetic stand for 5 minutes 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.
?. [Cleanup of PCR Amplification]
After the solution is clear (about 5 minutes), carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets (Caution do not discard beads).
?. [Cleanup of PCR Amplification]
Wash #1: 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.
?. [Cleanup of PCR Amplification]
Wash #2: Repeat the previous 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.
?. [Cleanup of PCR Amplification]
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 target. 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.
?. [Cleanup of PCR Amplification]
Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 15 μl of 0.1X TE. Mix well by pipetting up and down 10 times, or on a vortex mixer. Incubate for at least 2 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.
?. [Cleanup of PCR Amplification]
Place the tube/plate on the magnetic stand. After 5 minutes (or when the solution is clear), transfer 13 μl to a new PCR tube for and store at -20ºC.
?. [QC of libraries]
Check the size distribution on an Agilent Bioanalyzer® High Sensitivity DNA chip or TAPESTATION. The sample may need to be diluted before loading.
The size should be 160-350bp.Since the size of library is so near to the PCR dimer (128 bp), sometimes there are residual dimers. The ratio of dimers is mostly 1-20% in my experiments (please see attached screenshots of Tapestation analysis of two libraries). You can easily get rid of them by doing another round of 1.2X AMPure XP beads clean up.If you get too many dimer contamination, I recommend doing gel based size selection using a 2-3% agarose gel. Pippin Prep is a good option to do gel based size selection. Also consider reduce the Adaptor to as low as 3X when you make libraries for similar experiments in future.
1.2X AMPure XP purification can completely remove all dimers in this sample↑.This library needs gel based purification to remove dimers.
?. [Sequencing and data processing tips:]
Be sure to do pair end sequencing. This will give you the information of fragment size.Fragments that are 140 bp (library size >270 bp) contain surrounding nucleosome positioning information.
?. [Sequencing and data processing tips:]
Be aware that there will be several bases of adapter sequences in some of the reads, known as read through adaptors. For example, I sequence 42 bp each read. For all fragments that are shorter than 42bp, there will be adapter sequences at 3’ of the reads. These adaptor read through should be trimmed. My collaborator Qian Zhu has developed an easy to use pipeline CUT&RUNTools, please feel free to email Qian for any questions.