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DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
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DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795

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  1. ChIP
Chromatin Immunoprecipitation Image 1: DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
Figure 1. Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 50 ng, 5 ng, or 0.5 ng enriched ChIP DNA or 50 ng Input DNA using SimpleChIP® ChIP-seq Multiplex Oligos for Illumina (Dual Index Primers) #47538, pooled into one sample, and sequenced on an Illumina Next-Seq platform. The figure shows binding across GAPDH, a known target gene of H3K4me3. For additional ChIP-seq tracks, please download the product data sheet.
Chromatin Immunoprecipitation Image 2: DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
Figure 2. Chromatin immunoprecipitations were performed with cross-linked chromatin from 4 x 106 HCT 116 cells and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared from 50 ng, 5 ng, or 0.5 ng enriched ChIP DNA or 50 ng Input DNA using SimpleChIP® ChIP-seq Multiplex Oligos for Illumina (Dual Index Primers) #47538, pooled into one sample, and sequenced on an Illumina Next-Seq platform. The figure shows binding across CaMK2D, a known target gene of TCF4/TCF7L2. For additional ChIP-seq tracks, please download the product data sheet.
Chromatin Immunoprecipitation Image 3: DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
Figure 3. Metagene analysis of ChIP-seq data generated using different amounts of starting ChIP DNA. Analyses of both Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (left) and TCF4/TCF7L2 (C48H11) Rabbit mAb #2569 (right) ChIP-seq data show that the signal-to-noise ratio for peaks identified across entire genome are similar for all three starting amounts of ChIP DNA.
Chromatin Immunoprecipitation Image 4: DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
Figure 4. Agilent Bioanalyzer profiles of DNA libraries prepared from different starting amounts of ChIP DNA generated using the SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared from 50 ng (A), 5 ng (B), and 0.5 ng (C) of enriched ChIP DNA and 50 ng (D) input DNA. As shown, each DNA library preparation shows a similar DNA fragment size (expected range 300 bp to 900 bp).
Product Image 1: DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
Figure 5. CUT&RUN NGS data generated using Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and 3.6 ng of CUT&RUN DNA, as described in Table 2. The figure shows binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K4me3.
Product Image 2: DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
Figure 6. CUT&RUN NGS data generated using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569 and 0.7 ng of CUT&RUN DNA, as described in Table 2. The figure shows binding across chromosome 8 (upper), including MYC (lower), a known target gene of TCF4/TCF7L2.
Product Image 3: DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
Figure 7. CUT&RUN NGS data generated using Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499 and 3.1 ng of CUT&RUN DNA, as described in Table 2. The figure shows binding across chromosome 7 (upper), including ACTB (lower), a known target gene of Phospho-Rpb1.
Product Image 4: DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)
Figure 8. Agilent Bioanalyzer® profiles of DNA libraries prepared from different starting amounts of CUT&RUN DNA generated using the Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751. DNA libraries were prepared from 1 ng (A), 0.5 ng (B), 0.3 ng (C), and 0.1 ng (D) of enriched CUT&RUN DNA. As shown, each DNA library preparation shows a similar DNA fragment size.
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To Purchase # 56795
Cat. # Size Qty. Price
56795S		 1 Kit  (24 assays)
$ 750

Custom Formulation

Product Includes Volume (with Count) Storage Temp
End Prep Enzyme Mix 1 x 72 µl -20°C
End Prep Reaction Buffer 1 x 168 µl -20°C
Ligation Enhancer 1 x 24 µl -20°C
Ligation Master Mix 1 x 720 µl -20°C
Q5® PCR Master Mix 1 x 600 µl -20°C

Storage

Store all components at -20ºC. This product is stable for 12 months if stored properly.

Protocol

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DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN)

Next generation sequencing (NG-seq) is a high throughput method that can be used downstream of chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assays to identify and quantify target DNA enrichment across the entire genome. The DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) contains all of the enzymes and buffers necessary to generate high quality DNA sequencing libraries from ChIP DNA or CUT&RUN DNA for next-generation sequencing on the Illumina® platform. The fast, user-friendly workflow minimizes hands-on time needed for generation and purification of DNA libraries.

Each kit component must pass rigorous quality control standards, and for each new lot the entire set of reagents is functionally validated together by construction and sequencing of indexed libraries on the Illumina® sequencing platform.

This product must be used in combination with Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538. This product provides sufficient amounts of reagents for 24 reactions and is compatible with both enzymatic- or sonication-fragmented, ChIP-enriched DNA, and CUT&RUN DNA (with different protocols for DNA enriched from ChIP or CUT&RUN assays).

Compatible Assay kits:
SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003
SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005
SimpleChIP® Plus Sonication Chromatin IP Kit #56383
Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580
Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538
CUT&RUN Assay Kit #86652

Non-Compatible SimpleChIP® kits:
SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) #9002
SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004
Note: Agarose beads are blocked with sonicated salmon sperm DNA, which will contaminate DNA library preps and NG-seq.

Required Reagents
Reagents Included:

  1. • (green) End Prep Enzyme Mix #73416
  2. • (green) End Prep Reaction Buffer #93209
  3. • (red) Ligation Master Mix #46719
  4. • (red) Ligation Enhancer #30147
  5. • (blue) Q5® PCR Master Mix (2X) #67488

Reagents Not Included:

  1. Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538
  2. Nuclease-free Water #12931
  3. AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317)
  4. 80% Ethanol (freshly prepared)
  5. 1X TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
  6. 10 mM Tris-HCl (pH 8.0-8.5)
  7. Magnetic rack/stand
  8. Bioanalyzer® and Agilent High Sensitivity DNA Kit (Agilent Technologies, Inc.)
  9. PCR tubes and PCR Machine

DNA Library Prep Kit for Illumina ® Protocol for ChIP DNA

SAFE STOP This is a safe stopping point in the protocol, if stopping is necessary.

I. End Preparation

When performing ChIP-seq, it is necessary to generate a control DNA sequencing library that can be used to determine any experimental bias in DNA enrichment that is introduced during the ChIP assay and DNA library preparation. DNA purified from input chromatin (i.e. the chromatin that was used for the IP) is typically used to generate the control DNA library.

Starting Material: 500 pg –1 μg of ChIP DNA. To ensure optimal diversity of the DNA sequencing libraries, we recommend using 5 ng of ChIP-enriched DNA for transcription factor or co-factor ChIP-seq, 50 ng of ChIP-enriched DNA for total histone or histone modification ChIP-seq, and 50 ng of input DNA for the control DNA sequencing library. If necessary, less than 5 ng of ChIP-enriched DNA can be used for library generation; however, this may result in a lower diversity of library due to PCR bias during amplification.

Before starting:

  • Thaw End Prep Enzyme Mix (•) at room temperature.
  • Prepare 1X TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).
  1. Prepare 0.5-50 ng of ChIP DNA and relative input DNA (control DNA) in a sterile nuclease-free tube. Add 1X TE to generate a final volume of 50 μl for each DNA sample.
  2. Add 3 μl of End Prep Enzyme Mix (•) and 7 μl of End Prep Reaction Buffer (•) to each DNA sample to make a total reaction volume of 60 μl.
  3. Thoroughly mix the reactions by pipetting up and down at least 10 times and perform a quick spin to collect all liquid from the sides of the tube. The presence of a small amount of bubbles will not interfere with performance.
  4. Place in a thermocycler and run the following program:
    • 30 minutes at 20 °C
    • 30 minutes at 65 °C
    • Hold at 4 °C
  5. Proceed to Adaptor Ligation (Section II). 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.

II. Adaptor Ligation

Before starting:

  • Both Adaptor for Illumina® (•) and USER™ enzyme (•) can be found in either Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538.
  • Thaw Adaptor for Illumina® (•) at room temperature.
  • Mix the Ligation Master Mix by pipetting up and down several times.
  • Prepare approximately 2.5 μl of 10 mM Tris-HCl (pH 8.0-8.5) for each sample.
  1. Dilute the Adaptor for Illumina® (•) in 10 mM Tris-HCl (pH 8.0-8.5). If starting DNA is 5 ng to 100 ng, dilute the adaptor 1:10 to generate a working concentration of 1.5 mM. If starting DNA is less than 5 ng, dilute the adaptor 1:25 to generate a working concentration of 0.6 mM.
  2. Add 30 μl Ligation Master Mix (•), 1 μl Ligation Enhancer (•), and 2.5 μl diluted Adaptor for Illumina® directly to the 60 μl End Prep Reaction Mixture from step 5 in Section I.
    Note: Preparing a reaction premix containing the diluted adaptor ahead of time is NOT recommended.
  3. Thoroughly mix the ligation reaction by pipetting up and down at least 10 times and perform a quick spin to collect all liquid from the sides of the tube. The 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.
  4. Incubate at room temperature for 15 minutes.
  5. Add 3 μl of USER™ Enzyme (•) to the ligation mixture. Mix well and incubate at 37°C for 15 minutes with the heated lid set to ≥ 47°C.
  6. Proceed to Cleanup of Adaptor-ligated ChIP DNA (Section III). (Safe Stop) Alternatively, samples can be stored overnight at -20°C.

III. Cleanup of Adaptor-ligated ChIP DNA without Size Selection

Size selection is NOT recommended during the Cleanup Adaptor-ligated ChIP DNA phase, because it results in a dramatic decrease in both yield and diversity of ChIP-seq DNA libraries.

Before starting:

  • If using AMPure® XP beads, allow the beads to warm to room temperature for at least 30 minutes before use.
  • Resuspend AMPure® XP Beads or SPRIselect® beads by tube inversion or pipetting up and down.
  • Prepare 400 μl of 80% ethanol for each sample.
  • Prepare 17 μl of 10 mM Tris-HCl (pH 8.0-8.5) for each sample.
  1. Add 87 μl (0.9X) resuspended AMPure® XP beads or SPRIselect® beads to each adaptor ligation 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.
  2. Incubate samples on bench top for at least 5 minutes at room temperature.
  3. Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
  4. Once the solution is clear, carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
  5. 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.
  6. Repeat step 5 once for a total of two washes. Be sure to remove all visible liquid after the second wash.
  7. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
    Note: Do not over-dry the beads. This may result in lower recovery of DNA targets. Elute the samples when the beads are still glossy looking, but when all visible liquid has evaporated. When the beads start to crack, they are too dry.
  8. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 μl of 10 mM Tris-HCl (pH 8.0-8.5) per sample. Mix well by pipetting up and down 10 times. Incubate for at least 2 minutes at room temperature.
  9. Place the tube/plate on the magnetic stand and wait for 5 minutes. Once the solution is clear, transfer 15 μl of supernatant containing the DNA targets to a new PCR tube. Proceed to PCR Enrichment of Adaptor-ligated ChIP-DNA (Section IV). (Safe Stop) Alternatively, samples can be stored at -20°C.

IV. PCR Enrichment of Adaptor-ligated ChIP DNA

Before starting:

  • Universal PCR Primer for Illumina® (•) and twelve Index Primers for Illumina® (•) are provided in Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580. For use with Single Index Primers (#29580), use only one Index Primer per PCR reaction. Refer to #29580 data sheet for valid barcode combinations and tips for setting up PCR reactions.
  • Eight Index 5 Primers for Illumina® (white caps) and twelve Index 7 Primers for Illumina® (orange caps) are provided in Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538. For use with Dual Index Primers (#47538), use only one Index 5 primer and one Index 7 primer per reaction. Refer to #47538 data sheet for valid barcode combinations and tips for setting up PCR reactions.
  • Thaw primers and the purified adaptor-ligated ChIP DNA fragments (from step 9, Section III) at room temperature.
  1. Add the following components to a sterile PCR tube:
    Reagents Volume for 1 PCR Reaction (50 μl)
    Purified adaptor ligated ChIP-DNA fragments (from step 9, Section III) 15 μl
    Q5® PCR Master Mix (•) 25 μl
    Single Index Primer for Illumina® (•) (or Dual Index 7 Primer for Illumina® [orange cap] for dual indexing) 5 μl
    Universal PCR Primer for Illumina® (•) (or Dual Index 5 Primer for Illumina® [white cap] for dual indexing) 5 μl
  2. Thoroughly mix the reaction by pipetting up and down 10 times and perform a quick spin to collect all liquid from the sides of the tube.
  3. Place the tube on a thermo cycler and perform PCR amplification using the following PCR cycling conditions:
    a. Initial Denaturation 98°C 30 sec
    b. Denaturation 98°C 10 sec
    c. Anneal and Extension 65°C 15 sec
    d.

    For starting material of 50 ng ChIP DNA, repeat steps b and c for a total of 6 cycles.
    For starting material of 5 ng ChIP DNA, repeat steps b and c for a total of 10 cycles.
    For starting material of 0.5 ng ChIP DNA, repeat steps b and c for a total of 13 cycles.

    e. Final Extension 65°C 3 min
    f. Hold 4°C
  4. Proceed to Cleanup of PCR Amplification (Section V). (Safe Stop) Alternatively, samples can be stored at -20°C.

V. Cleanup of PCR Amplification

Before starting:

  • If using AMPure® XP beads, allow the beads to warm to room temperature for at least 30 minutes before use.
  • Resuspend AMPure® XP Beads or SPRIselect® beads by tube inversion or pipetting up and down.
  • Prepare 400 μl of 80% ethanol for each sample.
  • Prepare approximately 40 μl of 10 mM Tris-HCl (pH 8.0-8.5) for each sample.
  1. Add 45 μl (0.9X) resuspended AMPure® XP beads or SPRIselect® beads to 50 μl PCR reaction from step 4 in Section IV. 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.
  2. Incubate samples on bench top for at least 5 minutes at room temperature.
  3. Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
  4. Carefully remove and discard the supernatant. Be careful not to disturb the beads that contain DNA targets.
  5. 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.
  6. Repeat Step 5 once for a total of two washes. Be sure to remove all visible liquid after the second wash.
  7. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
    Note: Do not over-dry the beads. This may result in lower recovery of DNA targets. Elute the samples when the beads are still glossy looking, but when all visible liquid has evaporated. When the beads start to crack, they are too dry.
  8. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 μl of 10 mM Tris-HCl (pH 8.0-8.5) per sample. Mix well by pipetting up and down 10 times. Incubate for at least 2 minutes at room temperature.
  9. Place the tube/plate on the magnetic stand and wait for 5 minutes. Carefully transfer 30 μl of supernatant containing the DNA targets to a new tube. (Safe Stop) Libraries can be stored at -20°C.
  10. Measure the concentration of library DNA by Nanodrop or Picogreen assay. The concentration of the DNA library should be in the range of 10-40 ng/μl.
  11. Dilute 1 μl of the library DNA with 10mM Tris-HCl to a final concentration of 5-10 ng/μl. Use the diluted library DNA to determine size distribution using an Agilent Bioanalyzer® High Sensitivity DNA chip, according to the manufacturer’s instructions.
  12. If adaptor dimer (~128 bp for Single Index Primers or ~146 bp for Dual Index Primers) contamination is observed, repeat cleanup steps 1-10. Residual adaptors and/or adaptor dimers will strongly contaminate the sequencing reaction.
  13. Dilute final purified library samples with 10mM Tris-HCl for high throughput sequencing. Refer to Illumina® sequencing manual for optimal concentration and volume of library DNA required for NG-seq.

DNA Library Prep Kit for Illumina ® Protocol for CUT&RUN DNA

SAFE STOP This is a safe stopping point in the protocol, if stopping is necessary.

I. End Preparation

When performing CUT&RUN-seq, it is necessary to generate a control DNA sequencing library that can be used to determine any experimental bias in DNA enrichment that is introduced during the CUT&RUN assay and DNA library preparation. DNA purified from input DNA (refer to Section V of CUT&RUN Assay Kit #86652 Protocol) is typically used to generate the control DNA library.

Starting Material: 0.5 ng–1 µg of CUT&RUN DNA. The typical yield for CUT&RUN DNA is 0.6 to 6 ng per reaction with 100,000 starting cells. To ensure optimal diversity of the DNA sequencing libraries, we recommend using as much CUT&RUN DNA as you can obtain from a CUT&RUN reaction. If necessary, as little as 0.1 ng of CUT&RUN DNA can be used for library generation; however, this may result in a lower diversity of library due to PCR bias during amplification. For the control DNA sequencing library using input DNA, 5 - 10 ng is a good starting point. Please note that a Picogreen assay is needed to determine the concentration of CUT&RUN DNA because Nanodrop alone is not sensitive enough.

Before starting:

  • Thaw the End Prep Reaction Buffer #93209 ( •) at room temperature. Vortex if DTT is precipitated.
  • Prepare 1X TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).
  1. Prepare 0.5-10 ng of CUT&RUN DNA and relative input DNA (control DNA) in separate sterile nuclease-free tubes. Add 1X TE to generate a final volume of 50 μl for each DNA sample.
  2. Add 3 μl of End Prep Enzyme Mix ( •) and 7 μl of End Prep Reaction Buffer ( •) to each DNA sample to make a total reaction volume of 60 μl.
  3. Thoroughly mix the reactions by pipetting up and down at least 10 times and perform a quick spin to collect all liquid from the sides of the tube. The presence of a small amount of bubbles will not interfere with performance.
  4. Place in a thermocycler and run the following program:
    • 30 minutes at 20 °C
    • 30 minutes at 50 °C
    • Hold at 4 °C
  5. Proceed to Adaptor Ligation (Section II). 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.

II. Adaptor Ligation

Before starting:

  • Both Adaptor for Illumina ® ( •) and USER ™ enzyme ( •) can be found in either Multiplex Oligos for Illumina ® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina ® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538.
  • Thaw Adaptor for Illumina ® ( •) at room temperature.
  • Mix the Ligation Master Mix by pipetting up and down several times.
  • Prepare approximately 2.5 μl of 10 mM Tris-HCl (pH 8.0-8.5) for each sample.
  1. Dilute the Adaptor for Illumina ® ( •) in 10 mM Tris-HCl (pH 8.0-8.5) as indicated in the table to avoid the contamination of library DNA by excess adaptors.
    Starting DNA Adaptor Dilution Working Adaptor Concentration
    100 ng – 1 μg No Dilution 15 μM
    5 ng – 100 ng 1:10 1.5 μM
    2.5 ng – 5 ng 1:25 0.6 μM
    1.25 ng – 2.5 ng 1:50 0.3 μM
    Less than 1.25 ng 1:125 0.12 μM
  2. Add 30 μl Ligation Master Mix ( •), 1 μl Ligation Enhancer ( •), and 2.5 μl diluted Adaptor for Illumina ® directly to the 60 μl End Prep Reaction Mixture from step 5 in Section I.
    Note: Preparing a reaction premix containing the diluted adaptor ahead of time is NOT recommended. However, a premix of Ligation Master Mix and Ligation Enhancer is allowed if placed at 4°C for < 8 hours.
  3. Thoroughly mix the ligation reaction by pipetting up and down at least 10 times and perform a quick spin to collect all liquid from the sides of the tube. The 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.
  4. Incubate at room temperature for at least 15 minutes.
  5. Add 3 μl of USER ™ Enzyme ( •) to the ligation mixture. Mix well and incubate at 37°C for 15 minutes with the heated lid set to ≥ 47°C.
  6. Proceed to Cleanup of Adaptor-ligated CUT&RUN DNA (Section III). (Safe Stop) Alternatively, samples can be stored overnight at -20°C.

III. Cleanup of Adaptor-ligated CUT&RUN DNA without Size Selection

Size selection is NOT recommended during the Cleanup Adaptor-ligated CUT&RUN DNA phase, because it results in a dramatic decrease in both yield and diversity of DNA libraries.

Before starting:

  • If using AMPure ® XP beads, allow the beads to warm to room temperature for at least 30 minutes before use.
  • Resuspend AMPure ® XP Beads or SPRIselect ® beads by tube inversion or pipetting up and down.
  • Prepare 400 μl of 80% ethanol for each sample.
  • Prepare 17 μl of 10 mM Tris-HCl (pH 8.0-8.5) for each sample.
  1. Add 106 μl (1.1X) resuspended AMPure ® XP beads or SPRIselect ® beads to each adaptor ligation 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.
  2. Incubate samples on bench top for at least 5 minutes at room temperature.
  3. Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
  4. Once the solution is clear, carefully remove and discard the supernatant. Be careful to remove all liquid residues but not to disturb the beads that contain DNA targets.
  5. 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.
  6. Repeat step 5 once for a total of two washes. Be sure to remove all visible liquid after the second wash.
  7. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
    Note: Do not over-dry the beads. This may result in lower recovery of DNA targets. Elute the samples when the beads are still glossy looking, but when all visible liquid has evaporated. If the beads start to crack, they are too dry.
  8. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 17 μl of 10 mM Tris-HCl (pH 8.0-8.5) per sample. Mix well by pipetting up and down 10 times. Incubate for at least 2 minutes at room temperature.
  9. Place the tube/plate on the magnetic stand and wait for 5 minutes. Once the solution is clear, transfer 15 μl of supernatant containing the DNA targets to a new PCR tube. Proceed to PCR Enrichment of Adaptor-ligated ChIP-DNA (Section IV). (Safe Stop) Alternatively, samples can be stored at -20°C.

IV. PCR Enrichment of Adaptor-ligated CUT&RUN DNA

Before starting:

  • Universal PCR Primer for Illumina ® ( •) and twelve Index Primers for Illumina ® ( •) are provided in Multiplex Oligos for Illumina ® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580. For use with Single Index Primers (#29580), use only one Index Primer per PCR reaction. Refer to #29580 data sheet for valid barcode combinations and tips for setting up PCR reactions.
  • Eight Index 5 Primers for Illumina ® (white caps) and twelve Index 7 Primers for Illumina ® (orange caps) are provided in Multiplex Oligos for Illumina ® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538. For use with Dual Index Primers (#47538), use only one Index 5 primer and one Index 7 primer per reaction. Refer to #47538 data sheet for valid barcode combinations and tips for setting up PCR reactions.
  • Thaw primers and the purified adaptor-ligated CUT&RUN DNA fragments (from step 9, Section III) at room temperature.
  1. Add the following components to a sterile PCR tube:
    Reagents Volume for 1 PCR Reaction (50 μl)
    Purified adaptor ligated CUT&RUN-DNA fragments (from step 9, Section III) 15 μl
    Q5 ® PCR Master Mix ( •) 25 μl
    Single Index Primer for Illumina ® ( •) (or Dual Index 7 Primer for Illumina ® [orange cap] for dual indexing) 5 μl
    Universal PCR Primer for Illumina ® (•) (or Dual Index 5 Primer for Illumina ® [white cap] for dual indexing) 5 μl
  2. Thoroughly mix the reaction by pipetting up and down 10 times and perform a quick spin to collect all liquid from the sides of the tube.
  3. Place the tube on a thermo cycler and perform PCR amplification using the following PCR cycling conditions:
    a. Initial Denaturation 98°C for 30 sec
    b. Denaturation 98°C for 10 sec
    c. Anneal and Extension 65°C for 13 sec
    d. For 50 - 100 ng starting CUT&RUN DNA Repeat steps b and c for a total of 6 - 7 cycles
    For 5 - 50 ng starting CUT&RUN DNA Repeat steps b and c for a total of 8-12 cycles
    For 1 - 5 ng starting CUT&RUN DNA Repeat steps b and c for a total of 13 - 15 cycles
    For 0.5 - 1 ng starting CUT&RUN DNA Repeat steps b and c for a total of 16 - 17 cycles
    For 0.2 – 0.5 ng starting CUT&RUN DNA Repeat steps b and c for a total of 18 - 19 cycles
    For 0.1 -0.2 ng starting CUT&RUN DNA Repeat steps b and c for a total of 20 cycles
    e. Final Extension 65°C for 3 min
    f. Hold 4°C

     

  4. Proceed to Cleanup of PCR Amplification (Section V). (Safe Stop) Alternatively, samples can be stored at -20°C.

V. Cleanup of PCR Amplification

Before starting:

  • If using AMPure ® XP beads, allow the beads to warm to room temperature for at least 30 minutes before use.
  • Resuspend AMPure ® XP Beads or SPRIselect ® beads by tube inversion or pipetting up and down.
  • Prepare 400 μl of 80% ethanol for each sample.
  • Prepare approximately 40 μl of 10 mM Tris-HCl (pH 8.0-8.5) for each sample.
  1. Add 50 μl (1.0X) resuspended AMPure ® XP beads or SPRIselect ® beads to 50 μl PCR reaction from step 4 in Section IV. 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.
  2. Incubate samples on bench top for at least 5 minutes at room temperature.
  3. Place the tube/plate on an appropriate magnetic stand for 5 minutes to separate the beads from the supernatant.
  4. Carefully remove and discard the supernatant. Be careful to remove all liquid residues but not to disturb the beads that contain DNA targets.
  5. 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.
  6. Repeat Step 5 once for a total of two washes. Be sure to remove all visible liquid after the second wash.
  7. Air dry the beads for up to 5 minutes while the tube/plate is on the magnetic stand with the lid open.
    Note: Do not over-dry the beads. This may result in lower recovery of DNA targets. Elute the samples when the beads are still glossy looking, but when all visible liquid has evaporated. If the beads start to crack, they are too dry.
  8. Remove the tube/plate from the magnetic stand. Elute the DNA target from the beads by adding 33 μl of 10 mM Tris-HCl (pH 8.0-8.5) per sample. Mix well by pipetting up and down 10 times. Incubate for at least 2 minutes at room temperature.
    Note: A smaller volume of Tris-HCl (pH 8.0-8.5) (eg. 17 µl) can be used, if a higher concentration of library DNA is desired.
  9. Place the tube/plate on the magnetic stand and wait for 5 minutes. Carefully transfer 30 μl of supernatant containing the DNA targets to a new tube. (Safe Stop) Libraries can be stored at -20°C.
    Note: Transfer only 15 µl supernatant containing the DNA targets to a new tube if 17 µl of Tris-HCl (pH 8.0-8.5) is used in Step 8.
  10. Measure the concentration of library DNA by Nanodrop or Picogreen assay. The concentration of the DNA library should be in the range of 10-40 ng/μl.
  11. Dilute 1 μl of the library DNA with 10mM Tris-HCl to a final concentration of 5-10 ng/μl. Use the diluted library DNA to determine size distribution using an Agilent Bioanalyzer ® High Sensitivity DNA chip, according to the manufacturer’s instructions.
  12. If adaptor dimer (~128 bp for Single Index Primers or ~146 bp for Dual Index Primers) contamination is observed, repeat cleanup steps 1-10. Residual adaptors and/or adaptor dimers will strongly contaminate the sequencing reaction.
  13. Dilute final purified library samples with 10mM Tris-HCl for high throughput sequencing. Refer to Illumina ® sequencing manual for optimal concentration and volume of library DNA required for NG-seq.

APPENDIX A: Quality Control of the Kit Components

I. End Prep Enzyme Mix (•)

Description
End Prep Enzyme Mix is optimized to convert 500 pg-1 μg of fragmented DNA to repaired DNA having 5´-phosphorylated, 3´-dA-tailed ends.

Quality Control Assays

  1. SDS-PAGE Purity: SDS-PAGE analysis of each individual enzyme indicates > 95% enzyme purity.
  2. Endonuclease Activity: Incubation of a minimum of 10 μl of this enzyme mix with 1 μg of φX174 RF I DNA in assay buffer for 4 hours at 37°C in 50 μl reactions results in less than 10% conversion to RF II as determined by agarose gel electrophoresis.
  3. Phosphatase Activity: Incubation of a minimum of 10 μl of this enzyme mix in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl2) containing 2.5 mM p-nitrophenyl phosphate at 37°C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm.
  4. Functional Activity (Nucleotide Incorporation, Phosphorylation and dA-Tailing): 1 μl of this enzyme mix repairs and phosphorylates the ends of > 95% of 0.5ug of DNA fragments containing both 3´ and 5´ overhangs with 20 minutes at 25°C, in 1X End Repair Reaction buffer, as determined by capillary electrophoresis.

II. End Prep Reaction Buffer (•)

Quality Control Assays

  1. 16-Hour Incubation: 50 μl reactions containing this reaction buffer at 1X concentration and 1 μg of HindIII digested Lambda DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing this reaction buffer at 1X concentration and 1 μg T3 DNA incubated for 16 hours at 37°C also results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis.
  2. Endonuclease Activity: Incubation of this reaction buffer at a 1X concentration with 1 μg of φX174 RF I DNA for 4 hours at 37°C in 50 μl reactions results in less than 10% conversion to RF II as determined by agarose gel electrophoresis.
  3. RNase Activity: Incubation of this reaction buffer at 1X concentration with 40 ng of a FAM-labeled RNA transcript for 16 hours at 37°C results in no detectable RNase activity as determined by polyacrylamide gel electrophoresis.
  4. Phosphatase Activity: Incubation of this reaction buffer at a 1X concentration in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl2 ) containing 2.5 mM p-nitrophenyl phosphate at 37°C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm.

III. Ligation Master Mix (•)

Description
Ligation Master Mix is a ready-to-use solution of T4 DNA Ligase, proprietary ligation enhancer, and optimized reaction buffer.

Quality Control Assays

  1. 16-Hour Incubation: 50 μl reactions containing Ligation Master Mix at 1X concentration and 1 μg of HindIII digested Lambda DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing Ligation Master Mix at 1X concentration and 1 μg T3 DNA incubated for 16 hours at 37°C also results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis.
  2. Endonuclease Activity: Incubation of Ligation Master Mix at a 1X concentration with 1 μg of φX174 RF I DNA for 4 hours at 37°C in 50 μl reactions results in less than 10% conversion to RF II as determined by agarose gel electrophoresis.
  3. RNase Activity: Incubation of Ligation Master Mix at 1X concentration with 40 ng of a FAM-labeled RNA transcript for 16 hours at 37°C results in no detectable RNase activity as determined by polyacrylamide gel electrophoresis.
  4. Transformation Assay: LITMUS™ 28 vector is cut with EcoRV (blunt), treated with calf intestinal phosphatase and gel purified. Blunt inserts from a HaeIII digest of φX174 DNA are ligated into the vector at a 3:1 insert:vector ratio using the Ligation Master Mix Protocol. Ligation products are transformed as described. Each lot exceeds the following standards:

Efficiency (transformants/µg)

Recircularization Insertion
Blunt ends > 1 x 107 > 2.5 x 106
Uncut vector > 1 x 108 N/A

IV. Ligation Enhancer (•)

Quality Control Assays

  1. 16-Hour Incubation: 50 μl reactions containing 1 μl Ligation Enhancer and 1 μg of HindIII digested Lambda DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing 1 μl NEBNext 5´ SR Adaptor 3 and 1 μg of T3 DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis.
  2. Endonuclease Activity: Incubation of a 50 μl reaction containing 1 μl Ligation Enhancer with 1 μg of φX174 RF I supercoiled DNA for 4 hours at 37°C results in less than 10% conversion to RF II (nicked molecules) as determined by agarose gel electrophoresis.
  3. RNase Activity: Incubation of a 10 μl reaction containing 1 μl Ligation Enhancer with 40 ng of RNA transcript for 16 hours at 37°C resulted in no detectable degradation of RNA as determined by gel electrophoresis.
  4. Phosphatase Activity: Incubation of 1 μl Ligation Enhancer in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl2 ) containing 2.5 mM p-nitrophenyl phosphate at 37°C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm.

V. Q5® PCR Master Mix (2X) (•)

Description
The Q5® PCR Master Mix (2X) is specifically optimized for robust, high fidelity amplification of next-generation sequencing (NGS) libraries, regardless of GC content. The polymerase component of the master mix, Q5 High-Fidelity DNA Polymerase, is a novel thermostable DNA polymerase that possesses 3´→5´ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5 also has an ultra-low error rate (> 100-fold lower than that of Taq DNA Polymerase and ~12-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase). The buffer component of the master mix has been optimized for robust amplification, even with GC-rich amplicons and offers enhanced compatibility with a variety of beads used in typical NGS workflows. These features make the Q5® PCR Master Mix ideal for NGS library construction. This convenient 2X master mix contains dNTPs, Mg++ and a proprietary buffer, and requires only the addition of primers and DNA template for robust amplification. The inclusion of the hot start aptamer allows convenient room temperature reaction set up.

Quality Control Assays

  1. 16-Hour Incubation: A 50 μl reaction containing Q5® PCR Master Mix and 1 μg of HindIII digested λ DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis. 50 μl reactions containing Q5® PCR Master Mix and 1 μg of T3 DNA incubated for 16 hours at 37°C results in no detectable non-specific nuclease degradation as determined by agarose gel electrophoresis.
  2. Phosphatase Activity: Incubation of Q5® PCR Master Mix in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl2 ) containing 2.5 mM p-nitrophenyl phosphate at 37°C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm.
  3. Functional Activity (Multiplex PCR, Bead Inhibition): 30 cycles of PCR amplification of 20 ng genomic DNA with and without carboxylated magnetic beads in a 50 μl reaction containing 0.5 μM 4-plex primer mix and 1X Q5® PCR Master Mix result in the four expected amplicons and no inhibition of amplification in the presence of the beads.

posted November 2017

revised April 2022

protocol id: 1624

Protocol Id: 1624

Product Description

Next generation sequencing (NG-seq) is a high throughput method that can be used downstream of chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assays to identify and quantify target DNA enrichment across the entire genome. The DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) contains all of the enzymes and buffers necessary to generate high quality DNA sequencing libraries from ChIP or CUT&RUN DNA for next-generation sequencing on the Illumina® platform. The fast, user-friendly workflow minimizes hands-on time needed for generation and purification of DNA libraries. This product must be used in combination with Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538.



This product provides sufficient amounts of reagents for 24 reactions and is compatible with both enzymatic- or sonication-fragmented, ChIP-enriched DNA. Distinct protocols are provied for DNA library preparation from ChIP and CUT&RUN DNA. This product is compatible with SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003, SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, SimpleChIP® Plus Sonication Chromatin IP Kit #56383, and CUT&RUN Assay Kit #86652. This product is not compatible with SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) #9002 and SimpleChIP® Plus Enzymatic Chromatin IP Kit (Agarose Beads) #9004 because agarose beads are blocked with sonicated salmon sperm DNA, which will contaminate DNA library preps and NG-seq.

Specificity / Sensitivity

This kit has been validated in combination with Multiplex Oligos for Illumina® (Single Index Primers) (ChIP-seq, CUT&RUN) #29580 or Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538 to generate qualified DNA libraries using as little as 0.5 ng ChIP DNA or as little as 0.1 ng CUT&RUN DNA as starting materials.

Background

The chromatin immunoprecipitation (ChIP) assay is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1,2). This assay can be used to identify multiple proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome bound by a particular protein (3-6). It can be used to determine the specific order of recruitment of various proteins to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus (3,4). In addition to histone proteins, the ChIP assay can be used to analyze binding of transcription factors and co-factors, DNA replication factors and DNA repair proteins. When performing the ChIP assay, cells or tissues are first fixed with formaldehyde, a reversible protein-DNA cross-linking agent that "preserves" the protein-DNA interactions occurring in the cell (1,2). Cells are lysed and chromatin is harvested and fragmented using either sonication or enzymatic digestion. The chromatin is then immunoprecipitated with antibodies specific to a particular protein or histone modification. Any DNA sequences that are associated with the protein or histone modification of interest will co-precipitate as part of the cross-linked chromatin complex and the relative amount of that DNA sequence will be enriched by the immunoselection process. After immunoprecipitation, the protein-DNA cross-links are reversed and the DNA is purified. Standard PCR or Quantitative Real-Time PCR can be used to measure the amount of enrichment of a particular DNA sequence by a protein-specific immunoprecipitation (1,2). Alternatively, the ChIP assay can be combined with genomic tiling micro-array (ChIP on chip) techniques, high throughput sequencing, or cloning strategies, all of which allow for genome-wide analysis of protein-DNA interactions and histone modifications (5-8).
  1. Orlando, V. (2000) Trends Biochem Sci 25, 99-104.
  2. Kuo, M.H. and Allis, C.D. (1999) Methods 19, 425-33.
  3. Agalioti, T. et al. (2000) Cell 103, 667-78.
  4. Soutoglou, E. and Talianidis, I. (2002) Science 295, 1901-4.
  5. Mikkelsen, T.S. et al. (2007) Nature 448, 553-60.
  6. Lee, T.I. et al. (2006) Cell 125, 301-13.
  7. Weinmann, A.S. and Farnham, P.J. (2002) Methods 26, 37-47.
  8. Wells, J. and Farnham, P.J. (2002) Methods 26, 48-56.

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

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