May 14, 2023
CUT&RUN for nuclei using the CUTANA™ ChIC/CUT&RUN Kit
- 1Uppsala University
- methods
Protocol Citation: Eric RA Pederson 2023. CUT&RUN for nuclei using the CUTANA™ ChIC/CUT&RUN Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl85e78l2w/v1
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 10, 2021
Last Modified: May 14, 2023
Protocol Integer ID: 47152
Keywords: CUT&RUN, Nuclei, Frozen tissue
Abstract
Modified CUTANA™ ChIC/CUT&RUN Kit protocol using nuclei from brain tissue.
Guidelines
Taken from the Cutana ChIC/CUT&RUN Kit (https://www.epicypher.com/products/epigenetics-reagents-and-assays/cutana-chic-cut-and-run-kit) for more information.
Materials
NEXTFLEX Rapid DNA-Seq Kit 2.0 BundlePerkinElmerCatalog #NOVA-5188-12
KONTES Dounce Tissue GrindersKimble ChaseCatalog #KT885300-0002
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
HALT phosphatase and protease inhibitor cocktail (100x)Thermo Fisher ScientificCatalog #78442
2 ml LoBind TubesEppendorfCatalog #0030108078
1.5 mL LoBind tubes EppendorfCatalog #022431021
Cutana ChIC/CUT&RUN KitEpiCypherCatalog #14-1048
Roche Complete Protease Inhibitor EDTA-Free tablets Sigma AldrichCatalog #5056489001
Molecular Grade WaterATCCCatalog #60-2450
Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGe HealthcareCatalog #44152105050350
MACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458
NEBNext Ultra II DNA Library Prep Kit for IlluminaNEBCatalog #E7645S
NEBNext Ultra End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7442S
HEPESFisher ScientificCatalog #BP310
Triton X-100Sigma AldrichCatalog #T8787-50ML
Glycerol, 1000mlPromegaCatalog #H5433
Magnesium ChlorideFisher ScientificCatalog #AC223210010
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584
High Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585
Equipment
new equipment
NAME
Qubit 2.0 Fluorometer instrument
BRAND
Q33226
SKU
with Qubit RNA HS Assays
SPECIFICATIONS
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
LINK
Equipment
Thermomixer C
NAME
Eppendorf
BRAND
2231000667
SKU
LINK
| 10mM | Tris pH 8.0 | |
| 250mM | sucrose | |
| 25mM | KCl | |
| 5mM | MgCl2 | |
| 0.10% | Triton X-100 | |
| 0.50% | RNasin Plus RNase inhibitor Promega Catalog #N2611 | |
| 0.20% | 1X protease Inhibitor Promega Catalog #G6521 | |
| 0.1mM | DTT |
HB
Protocol materials
1.5 mL LoBind tubes EppendorfCatalog #022431021
KONTES Dounce Tissue GrindersKimble ChaseCatalog #KT885300-0002
NEBNext Ultra II DNA Library Prep Kit for IlluminaNEBCatalog #E7645S
NEBNext Ultra End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7442S
HEPESFisher ScientificCatalog #BP310
High Sensitivity D1000 ReagentsAgilent TechnologiesCatalog #5067-5585
HALT phosphatase and protease inhibitor cocktail (100x)Thermo Fisher ScientificCatalog #78442
2 ml LoBind TubesEppendorfCatalog #0030108078
MACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458
Triton X-100Merck MilliporeSigma (Sigma-Aldrich)Catalog #T8787-50ML
Magnesium ChlorideFisher ScientificCatalog #AC223210010
High Sensitivity D1000 ScreenTapeAgilent TechnologiesCatalog #5067-5584
Roche Complete Protease Inhibitor EDTA-Free tablets Merck MilliporeSigma (Sigma-Aldrich)Catalog #5056489001
Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350
Glycerol, 1000mlPromegaCatalog #H5433
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Cutana ChIC/CUT&RUN KitEpiCypherCatalog #14-1048
NEXTFLEX Rapid DNA-Seq Kit 2.0 BundlePerkinElmerCatalog #NOVA-5188-12
Molecular Grade WaterATCCCatalog #60-2450
NEXTFLEX Rapid DNA-Seq Kit 2.0 BundlePerkinElmerCatalog #NOVA-5188-12
2 ml LoBind TubesEppendorfCatalog #0030108078
1.5 mL LoBind tubes EppendorfCatalog #022431021
50 ml Falcon tube
MACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458
1X protease inhibitorPromegaCatalog #G6521
RNasin Plus RNase inhibitor PromegaCatalog # N2611
Roche Complete Protease Inhibitor EDTA-Free tablets Merck MilliporeSigma (Sigma-Aldrich)Catalog #5056489001
PMSFP212121Catalog #RP-P20270
HALT Protease and Phosphatase Inhibitor CocktailAbcamCatalog #78440
DTTP212121Catalog #SV-DTT
Safety warnings
Regular lab safety rules apply. Ensure the use of googles during the usage of dry ice and formaldehyde.
Before start
The pre-experimentation steps are important
Also it is important that all the primers have been ordered and reconstituted beforehand.
pre-experimentation
pre-experimentation
All steps should be performed on ice or at 4 °C .
1m
Pre-chill all Dounces and pestles to 4 °C in a fridge. Or just leave on ice for a while.
10m
Pre-chill all tubes.
For each sample you are processing, you will need:
(i) One 2 mL 2 ml LoBind TubesVWR InternationalCatalog #0030108078 per sample
(ii) Three 1.5 mL LoBind tubes VWR InternationalCatalog #022431021 per sample
(iii) one PCR tube per sample
(iv) One 50 ml Falcon tubeVWR International for filtration step per sample
Prepare buffers.
i) Homogenization buffer
ii) 25X protease inhibitor
iii) wash buffer (see step 5)
iv) CP buffer (see step 5)
v) antibody buffer (see step 5)
vi) Nuclear Buffer (see step 26)
Homogenization buffer (HB);
| A | B | |
| 10mM | Tris pH 8.0 | |
| 250mM | sucrose | |
| 25mM | KCl | |
| 5mM | MgCl2 | |
| 0.10% | Triton X-100 | |
| 0.50% | RNasin Plus RNase inhibitor Promega Catalog #N2611 | |
| 0.20% | 1X protease Inhibitor Promega Catalog #G6521 | |
| 0.1mM | DTT |
Homogenization buffer
RNasin Plus RNase inhibitor VWR InternationalCatalog # N2611
1X protease inhibitorVWR InternationalCatalog #G6521
ii) 25X protease inhibitor
1 protease inhibtor tablet (Roche) in 2 mL water (25X stock)
Store remaining 25X stock for 12 weeks at -20 °C
Roche Complete Protease Inhibitor EDTA-Free tablets VWR InternationalCatalog #5056489001
DAY 1 - Section I - Buffer Prep
DAY 1 - Section I - Buffer Prep
30m
30m
| A | B | C | D | E | F | G | H | I | |
| # of Samples | 1X | 2X | 3X | 4X | 5X | 6X | 8X | 16X | |
| Wash Buffer – | |||||||||
| Pre-wash buffer | 1.8 ml | 3.6 ml | 5.4 ml | 7.2 ml | 9 ml | 10.8 ml | 14.4 ml | 28.8 ml | |
| 25X Protease inhibitor | 72 ul | 144 ul | 216 ul | 288 ul | 360 ul | 432 ul | 576 ul | 1.15 ml | |
| 1 M Spermidine | 0.9 ul | 1.8 ul | 2.7 ul | 3.6 ul | 4.5 ul | 5.4 ul | 7.2 ul | 14.4 ul | |
| **1% Triton X-100 | 0.02 ul | 0.038 ul | 0.056 ul | 0.074 ul | 0.092 ul | 0.10 ul | 0.146 ul | 0.30 ul | |
| ** 0.05% SDS | 0.015 ul | 0.19 ul | 0.028 ul | 0.037 ul | 0.046 ul | 0.05 ul | 0.073 | 0.15 ul | |
| Cell Permeabilization buffer – | |||||||||
| Wash Buffer | 1.4 ml | 2.8 ml | 4.2 ml | 5.6 ml | 7 ml | 8.4 ml | 11.2 ml | 22.4 ml | |
| 5% Digitonin | 2.8 ul | 5.6 ul | 8.4 ul | 11.2 ul | 14 ul | 16.8 ul | 22.4 ul | 44.8 ul | |
| Antibody Buffer – | |||||||||
| Cell Permeabilization buffer | 100 ul | 200 ul | 300 ul | 400 ul | 500 ul | 600 ul | 800 ul | 1.6 ml | |
| 0.5 M EDTA | 0.4 ul | 0.8 ul | 0.16 ul | 0.32 ul | 0.64 ul | 0.128 ul | 3.2 ul | 6.4 ul |
buffer scaling calculations
Wash buffer - store at RT for use on Day 1
CP buffer - store at 4° for use on Day 2
Antibody buffer - store on ice for use on Day 1
Example of making solutions:
Add 1.8 mL of Pre-Wash Buffer per sample to a single conical tube labeled Wash Buffer
Add 72 µL μL of 25X protease inhibitor stock per sample to the Wash Buffer
Add 0.9 µL ul of 1M Spermindine per sample to the Wash Buffer
Room temperature
Transfer 1.4 mL of Wash Buffer per sample into a new conical tube labelled Cell Permeabilization Buffer
Add 2.8 µL of 5% digitonin to CP buffer
Store the remaining Cell Permeabilization Buffer at 4 °C Overnight (for Day 2 use).
Transfer 100 µL per sample of Cell Permeabilization Buffer into a new tube labelled “Antibody Buffer”
Add 0.4 µL 0.5 Molarity (M) EDTA per sample
Store final buffer on ice.
Section II - Bead Activation
Section II - Bead Activation
30m
30m
Gently resuspend the ConA Beads by pipetting.
Transfer 11 µL /sample to a 1.5 mL tube for batch processing.
*NOTE: Batch processing at this step is recommended to improve sample handling.
If a 1.5 mL tube magnet is not available, the beads can be processed individually (10 μL/ sample) in the provided 8-strip PCR tubes using a compatible 8-strip magnet.
Place the tube on a magnet until slurry clears and pipette to remove sup.
*IMPORTANT: For all steps involving magnetic racks, take care to avoid disturbing the immobilized beads with pipette tips.
To avoid drying the beads, immediately add 100 µL /sample cold Bead Activation Buffer.
Pipette gently to mix.
Place the tube on a magnet until slurry clears and pipette to remove sup.
Repeat previous step for total of two washes.
Resuspend beads in 11 µL /sample cold Bead Activation Buffer.
*NOTE: If not batch processing, use 10 μL/sample at this step. Proceed directly to Section III.
For each experimental condition, aliquot 10 µL /sample of activated bead slurry into separate 8-strip tubes.
Keep on ice until needed.
Tissue preparation and Sample Preparation: Nuclei
Tissue preparation and Sample Preparation: Nuclei
1h
1h
In the most sterile way possible, cut a small piece of tissue, around 50 mg, and leave it in the petri dish with a marking on the lid, in the dry ice. Weigh it and cut again if needed.
Make sure to use the ethanol to clean everything and be careful not to cut yourself.
Add 2 mL of 1X HB buffer into the dounce, which is sitting in the ice.
Place 20 mg frozen tissue into a pre-chilled 2 mL Dounce containing 2 mL cold 1x HB and let thaw for 00:05:00 .
Dounce with “A” loose pestle until resistance goes away (~10 strokes).
Put the A pestle into the beaker of water
Dounce with “B” tight pestle for 20 strokes.
Put the B pestle into the beaker of water
Pour everything from the dounce into a 30 um MACS smartstrainer which is sitting on top of a labelled 50 mL falcon tube sitting in ice.
MACS SmartStrainers 30umVWR InternationalCatalog #130-098-458
Let it drip through for00:15:00
15m
Transfer to a labelled lobind eppendorf tube, already cold from sitting in ice.
To pellet the nuclei, centrifuge 900 rpm, 4°C, 00:10:00
10m
Transfer the supernatant to a new tube without disturbing the pellet
Repeat the centrifugation
10m
Discard supernatant
Section III - Binding cells to activated beads
Section III - Binding cells to activated beads
30m
30m
| A | B | |
| Reagents | Amount | |
| HEPES buffer | 20 mM | |
| KCl (pH 7.9) | 10 mM | |
| Triton X-100 | 0.1% | |
| Glycerol | 20% | |
| MnCl2 | 1 mM |
Nuclear extraction buffer
Prepare 410 µL / sample (+ extra dead volume) Nuclear Extraction Buffer fresh the day of use. Sterile Filter.
Add 1:10,000 dilution of 1 Molarity (M) Spermindine and 1X Protease Inhibitor to the Nuclear Extraction Buffer. Place final buffer on ice.
200/10000 = 0.2 ul
Resuspend cells in 100 µL per sample 1X PBS.
Set aside 10 µL cells for future analysis by Trypan blue staining (intact cell control).
Centrifuge for 600 rpm, 4°C, 00:03:00 . Remove and discard supernatant
*NOTE:For all steps, the ratio of buffer volumes; cells scales linearly. For example, use 1 mL buffer for 5 x 10^6 cells.
3m
Resuspend cells in 200 µL per sample cold Nuclear Extraction Buffer.
Incubate 0 °C 10m (ice)
Centrifuge for 600 rpm, 4°C, 00:03:00
Remove and discard sup.
The pellet should change in appearance from sticky, pale yellow (cells) to white and fluffy (nuclei).
3m
Add 10 µL Trypan blue to the intact cell control (Step 4) and the isolated nuclei (previous step).
Load onto hemacytometer slide and examine under brightfield/phase microscope to determine whether nuclei have been efficiently isolated (Figure 12).
To cryopreserve nuclei, slowly freeze samples in isopropanol-filled chiller in -80 °C freezer.
It might be a good idea to add 300 µL of freezing medium to each tube for saving
| Hepes pH 7.5 | 10 mM | 1 ml | 1 M stock | |
| MgCl2 | 2 mM | 200 ul | 1 M stock | |
| KCl | 25 mM | 800 ul | 3 M stock | |
| H20 | 98 ml |
Hyporonic Buffer N;
Mix 15 mL of buffer N containing 5 µL Halt protease inhibitor cocktail, 1 millimolar (mM) DTT and 1 millimolar (mM) PMSF with 35 mL of pure glycerol
PMSFVWR InternationalCatalog #RP-P20270
DTTVWR InternationalCatalog #SV-DTT
HALT Protease and Phosphatase Inhibitor CocktailVWR InternationalCatalog #78440
When ready to use samples for CUT&RUN, thaw nuclei quickly by placing on 37 °C block.
Centrifuge down and reconstitute
Proceed to CUT&RUN ConA Bead conjugation step (Experimental Protocol, Step 10).
Resuspend cells in 105 µL /sample in RT Wash Buffer. Pipette to thoroughly resuspend.
Aliquot 100 µL washed cells to each 8-strip tube containing 10 µL of activated beads. Gently vortex or pipette to mix.
*NOTE: Beads are prone to clumping. If your beads are clumped, continue to vortex and/or pipette mix to ensure even resuspension.
Incubate cell-bead slurry on the platform rocker for 00:10:00 at Room temperature to adsorb cells to beads.
*NOTE: Count cells by Trypan staining prior to incubation with ConA beads. After incubation with ConA beads, check the sup to ensure most cells have adsorbed to the beads.
10m
I have been moving the tube to ice for 00:50:00 as an extra time incubation
50m
Sample Preparation: Cross-Linking (Optional)
Sample Preparation: Cross-Linking (Optional)
30m
30m
Labile targets or highly transient chromatin binding proteins may be improved by cross-linking
Transfer 500,000 cells or amount that you wish to use into a 1.5 mL lobind tube
Add formaldehyde directly to culture to achieve desired final concentration of formaldehyde.
(recommended 0.1%-1%)
I use 16% formaldehyde in 200 µL of nuclear extraction buffer, which requires 13.75 µL of formaldehyde.
Incubate Room temperature 10m on
Equipment
Bio RS-24 Mini-rotator
NAME
mini-rotator
TYPE
BioSan
BRAND
RS-24
SKU
LINK
10m
Quench the fixation by adding 2.5 Molarity (M) glycine to a final concentration of 125 millimolar (mM)
In this case it would be 11 µL
600 rpm, 4°C, 00:03:00
Discard supernatant
Section IV - ANTIBODY BINDING
Section IV - ANTIBODY BINDING
18h
18h
If using a multi-channel pipette (recommended) place a multi-channel reagent reservoir on ice.
*IMPORTANT: If processing > 8 samples (more than 1 x 8-strip tube), for subsequent wash steps remove & replace sups for a single strip before processing the next strip. This avoids bead dry out during wash steps.
*NOTE: Multi-channel pipetting is highly recommended through the rest of the experiment. This helps to avoid bead dry out, improves yield, and increases experimental throughput.
Place the 8-strip tubes on an 8-strip tube magnet (high volume setting) until slurry clears.
Pipette to remove sup, taking care to avoid disturbing the immobilized beads with pipette tip.
Immediately add 50 µL cold Antibody Buffer to each sample and gently vortex and/or pipette mix to prevent beads from drying.
(Optional)
Add 2 µL SNAP-CUTANA K-MetStat Panel per 500,000 cells.
The samples designated for the positive (H3K4me3) and negative (IgG) control antibodies.
Add 0.5 µg antibody to each sample and gently vortex.
Incubate 8-strip tubes on rotator Overnight at 4 °C .
DAY 2 - Section IV - Antibody binding
DAY 2 - Section IV - Antibody binding
10m
10m
If using a multi-channel pipette (recommended) place a multi-channel reagent reservoir on ice.
Fill with Cell Permeabilization Buffer.
*IMPORTANT: If processing > 8 samples (more than 1 x 8-strip tube), for subsequent wash steps remove & replace sups for a single strip before processing the next strip. This avoids bead dry out during wash steps.
Place the 8-strip tubes on magnet until slurry clears. Pipette to remove sup.
*While beads are on magnet*, add 200 µL cold Cell Permeabilization Buffer directly onto beads.
Pipette to remove supernatant
Repeat previous step for total of two washes, without removing 8-strip tubes from the magnet.
Add 50 µL cold Cell Permeabilization Buffer to each sample.
Gently vortex and/or disperse clumps by thorough pipetting.
Section V - BINDING OF PAG-MNASE
Section V - BINDING OF PAG-MNASE
30m
30m
Add 2.5 µL pAG-MNase (20x stock) to each sample.
Gently vortex/pipette mix.
*NOTE: To evenly distribute enzyme across cells/nuclei, ensure beads are thoroughly
resuspended by gentle pipetting with a P200.
Incubate samples for 00:10:00 at Room temperature on the platform shaker
Return 8-strip tube to magnet.
Remove sup.
10m
*While beads are on magnet*, add 200 µL cold Cell Permeabilization Buffer directly onto beads.
Pipette to remove sup.
Repeat previous step for total of two washes without removing 8-strip tubes from the magnet.
Remove 8-strip tubes from the magnet.
Add 50 µL cold Cell Permeabilization Buffer to each sample.
Gently vortex and disperse clumps by pipetting.
Cover/ put away Cell Permeabilization Buffer for later use.
Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
Section VI - TARGETED CHROMATIN DIGESTION AND RELEASE
3h
3h
Place 8-strip tubes on ice.
Add 3 µL Calcium Chloride to each sample and gently vortex.
Ensure efficient digestion by making sure beads are thoroughly resuspended.
Gently pipette with a P200 if needed.
Incubate 8-strip tubes on rotator for 02:00:00 at 4 °C .
2h
Add 33 µL Stop Buffer to each sample.
Gently vortex to mix.
Prior to first use, reconstitute E. coli Spike-in DNA in 200 µL DNase free water.
*IMPORTANT: Lyophilized DNA pellet is invisible to the eye.
Prior to opening, pellet DNA by quick spin in a benchtop microfuge.
After reconstitution, vortex tube on all sides to ensure complete resuspension.
Add 1-2 µL Spike-in DNA to each sample. Gently vortex to mix.
*NOTE: In general, aim for Spike-in DNA to comprise 0.5 – 5% (ideally closest to 1%) of total read counts in the sequencing data.
Therefore, while 0.5 ng is a good starting amount for both high (e.g. H3K27me3) and low (e.g. H3K4me3) abundance targets, this may need to be adjusted higher or lower depending on the antibody used, target of interest, total DNA yield, and sequencing results.
Incubate 8-strip tubes for 00:10:00 at 37 °C in a thermocycler.
10m
(Optional - if Cross-Linking extra methodology only)
Place 8-strip tubes on magnet stand until slurry clears.
Transfer supernatants containing DNA to new 8-strip tubes
Reverse cross-links by adding 0.8 µL 10% SDS and 1 µL of 20 µg/µL Proteinase K to each reaction.
Mix by vortexing
Overnight 55 °C
in a thermocycler
Quick spin in benchtop microfuge.
Place 8-strip tubes on a magnet stand until slurry clears.
Transfer sups containing CUT&RUN enriched DNA to 1.5 mL tubes and discard ConA Beads.
Section VII - DNA PURIFICATION
Section VII - DNA PURIFICATION
30m
30m
Add 420 µL DNA Binding Buffer to each sample.
Mix well by vortexing.
For every sample, place a DNA Cleanup Column into a DNA Collection Tube.
Load each sample onto a column and label the top.
Centrifuge for 16.000 x g, 00:00:30 Room temperature
Discard the flow-through.
Place the collection tube back on to the column.
*NOTE: A vacuum manifold can be used in place of centrifugation.
For each step, add the indicated buffer, turn the vacuum on, and allow the solution to pass through the column before turning the vacuum off.
30s
Prior to first use, add 20 mL ≥ 95% ethanol to DNA Wash Buffer.
Section VII (continued)
Section VII (continued)
SECTION VII: DNA PURIFICATION (~30 MIN), CONTINUED
Add 200 µL DNA Wash Buffer to each sample column
Centrifuge for16.000 x g, 00:00:30 , Room temperature
Discard the flow-through.
Place the collection tube back on to the column.
30s
Repeat for a total of two washes.
Discard the flow-through.
Centrifuge one additional time for 16.000 x g, 00:00:30 to completely dry the column.
30s
Transfer column to a clean pre-labeled 1.5 mL lobind tube, ensuring the column does not come into contact with the flow-through.
Elute DNA by adding 12 µL DNA Elution Buffer, taking care to ensure the buffer is added to the center of the column rather than the wall.
Tap the column + collection tube on the benchtop to ensure all droplets are absorbed onto the resin.
*NOTE: 12 μL is recommended, however DNA can be eluted in 6 – 20 μL volumes depending on anticipated yield and desired final concentration.
Larger elution volumes, longer incubation times, and/or multiple rounds of elution may improve DNA yield. However, sample concentration will be reduced with larger total elution volume.
Let sit00:05:00 , then centrifuge for 16.000 x g, 00:01:00 , RT.
6m
Vortex eluted material and use 1 µL to quantify the CUT&RUN-enriched DNA using the Qubit fluorometer as per the manufacturer’s instructions.
See Quality Control Checks section for typical DNA yields.
CUT&RUN DNA can be stored at -20 °C for future processing.
Section VIII - Library Preparation
Section VIII - Library Preparation
18m
18m
1) End Prep
Starting Material: 500 pg –1 µg fragmented DNA. We recommend that DNA be sheared in 1X TE
If the DNA volume post shearing is less than 50 µL , add 1X TE to a final volume of 50 µL
Alternatively, samples can be diluted with 10 millimolar (mM) Tris-HCl, pH 8.0 or 0.1X TE.
NEBNext End Prep
Add the following components to a sterile nuclease-free tube:
| A | B | |
| Reagent | Amount | |
| NEBNext Ultra II End Prep Enzyme Mix | 7 ul | |
| NEBNext Ultra II End Prep Reaction Buffer | 3 ul | |
| DNA | 50 ul | |
| Volume | 60 ul |
End Prep Reaction
Set a 100 µL or 200 µL pipette to 50 µL 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 thermocycler, with the heated lid set to ≥ 75 °C , and run the following program:
| A | B | |
| Time | Temperature | |
| 30 minutes | 20° | |
| 30 minutes | 65° | |
| Hold | 4° |
End Prep Incubation
If necessary, samples can be stored at –20°C; however, a slight loss in yield (~20%) may be observed. We recommend continuing with adaptor ligation before stopping.
Serapure purification
- Add 1.8X Speed beads to samples (108 µL beads to 60 µL reaction)
- Flick to mix
- Incubate for 00:10:00 at room temperature
- Use the magnet to collect the DNA-beads
- Discard supernatant
- Wash with 70% ethanol
- Dry at 37 °C for 00:03:00
- Elute with 20 µL TE buffer
- Incubate for 00:05:00 at room temperature
- Use the magnet to collect the DNA-beads
- Collect supernatant into a new tube
18m
Universal Adapter ligation
Add the following components directly to the End Prep Reaction Mixture:
| A | B | |
| Reagent | Amount | |
| End Prep Reaction Mixture | 20 | |
| NEBNext Quick T4 DNA Ligase | 1 ul | |
| NEBNext Ligation Buffer | 2.5 ul | |
| NextFlex unique adaptor | 2.5 ul | |
| Total Volume | 26 ul |
Universal Adapter ligation
NEXTFLEX Rapid DNA-Seq Kit 2.0 BundleVWR InternationalCatalog #NOVA-5188-12
Or one can skip the purification and run the ligation suggested in the NEB protocol
| A | B | |
| Reagent | Amount | |
| End Prep Reaction Mixture | 60 ul | |
| NEBNext Ligation Enhancer | 2.5 μl | |
| NEBNext Ligation Master Mix | 30 ul | |
| NEBNext Adaptor | 1 μl | |
| Total volume | 93.5 μl |
Universal Adapter ligation
Incubate the reaction for 00:10:00 at room temperature.
10m
Serapure purification
- Add 0.8X Speed beads to samples (20 µL Speed beads to 25 µL reaction)
- Flick to mix
- Incubate for 00:10:00 at room temperature
- Use the magnet to collect the DNA-beads
- Discard supernatant
- Wash with 70% ethanol
- Dry at 37 °C for 00:03:00
- Elute with 21 µL TE buffer
- Incubate for 00:05:00 at room temperature
- Use the magnet to collect the DNA-beads
- Collect supernatant into a new tube
18m
PCR and primer indexing
| A | B | |
| Reagent | Amount | |
| Next Flex primer mix | 5 ul | |
| NEBNext Master Mix | 25 ul | |
| Sample | 20 ul |
PCR and primer indexing according to the following cycling parameters:
| A | B | C | |
| Tempurature | Time | Cycle | |
| 98° | 45 seconds | 1 | |
| 98° | 15 seconds | 14 | |
| 60° | 10 seconds | - | |
| 72° | 1 minute | - | |
| 72° | 10 minutes | 1 | |
| 4° | hold | 1 |
Serapure purification
- Add 1.0X Speed beads to samples (50 µL Speed beads to 50 µL reaction)
- Flick to mix
- Incubate for 00:10:00 at room temperature
- Use the magnet to collect the DNA-beads
- Discard supernatant
- Wash with 70% ethanol
- Dry at 37 °C for 00:03:00
- Elute with 20 µL TE buffer
- Incubate for 00:05:00 at room temperature
- Use the magnet to collect the DNA-beads
- Collect supernatant into a new tube
18m
Qubit and Tapestation (D1000) analysis