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29580
Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN)
ChIP Kits & Reagents
ChIP Kit

Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN) #29580

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To Purchase # 29580
Cat. # Size Qty. Price
29580S
1 Kit  (24 assays)

Product Includes Volume (with Count) Storage Temp
Adaptor for Illumina Systems 1 x 240 µl -20°C
USER Enzyme 1 x 72 µl -20°C
Universal PCR Primer for Illumina Systems 1 x 120 µl -20°C
Index 1 Primer for Illumina Systems 1 x 10 µl -20°C
Index 2 Primer for Illumina Systems 1 x 10 µl -20°C
Index 3 Primer for Illumina Systems 1 x 10 µl -20°C
Index 4 Primer for Illumina Systems 1 x 10 µl -20°C
Index 5 Primer for Illumina Systems 1 x 10 µl -20°C
Index 6 Primer for Illumina Systems 1 x 10 µl -20°C
Index 7 Primer for Illumina Systems 1 x 10 µl -20°C
Index 8 Primer for Illumina Systems 1 x 10 µl -20°C
Index 9 Primer for Illumina Systems 1 x 10 µl -20°C
Index 10 Primer for Illumina Systems 1 x 10 µl -20°C
Index 11 Primer for Illumina Systems 1 x 10 µl -20°C
Index 12 Primer for Illumina Systems 1 x 10 µl -20°C

Storage

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

Protocol

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Multiplex Oligos for Illumina Systems (Single Index Primers) (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. Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN) contain adaptors and primers that are ideally suited for multiplex sample preparation for NG-seq on the Illumina Systems platform (Illumina, Inc.). This kit can be used to generate up to 12 distinct, barcoded ChIP-seq or CUT&RUN DNA libraries that can be combined into a single sequencing reaction.

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 Systems sequencing platform.

This product provides enough reagents to support up to 24 DNA sequencing libraries, and must be used in combination with the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795.

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
DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795
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. (red) Adaptor for Illumina Systems #42436
  2. (red) USER Enzyme #59713
  3. (blue) Universal PCR Primer for Illumina Systems #12078
  4. (blue) Index 1 Primer for Illumina® #28248
  5. (blue) Index 2 Primer for Illumina Systems #41836
  6. (blue) Index 3 Primer for Illumina Systems #64036
  7. (blue) Index 4 Primer for Illumina Systems #83765
  8. (blue) Index 5 Primer for Illumina Systems #18392
  9. (blue) Index 6 Primer for Illumina Systems #27180
  10. (blue) Index 7 Primer for Illumina Systems #43985
  11. (blue) Index 8 Primer for Illumina Systems #68962
  12. (blue) Index 9 Primer for Illumina Systems #83219
  13. (blue) Index 10 Primer for Illumina Systems #90275
  14. (blue) Index 11 Primer for Illumina Systems #28019
  15. (blue) Index 12 Primer for Illumina Systems #39090

Reagents Not Included:

  1. Enzymes and buffers appropriate for ChIP or CUT&RUN Illumina Systems NG-seq library preparation: provided in DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795
  2. Nuclease-free Water #12931
  3. AMPure XP Beads (Beckman Coulter, Inc. #A63881) or SPRIselect Reagent Kit (Beckman Coulter, Inc. #B23317)
  4. Freshly prepared 80% Ethanol
  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 Separation Rack #7017/#14654
  8. Agilent Bioanalyzer Systems and Agilent High Sensitivity DNA Kit (Agilent Technologies, Inc.)
  9. PCR tubes and PCR machine

Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN) Protocol

I. Low Plexity Pooling Guidelines:

Illumina Systems NG-seq platforms use a red laser/LED to sequence A/C and a green laser/LED to sequence G/T. For each cycle, both the red and the green channel need to be read to ensure proper image registration (i.e. A or C must be present in each cycle, and G or T must be present in each cycle). If this color balance is not maintained, sequencing the index read could fail. Please check the sequences of each index to be used to ensure that you will have signal in both the red and green channels for every cycle. See example below:

Index 7 Primers for Illumina Systems Index 5 Primers for Illumina Systems
Index 1 Primer for Illumina Systems

ATCACG

Index 1 Primer for Illumina Systems

ATCACG

Index 2 Primer for Illumina Systems

CGATGT

Index 2 Primer for Illumina Systems

CGATGT

Index 4 Primer for Illumina Systems

TGACCA

Index 3 Primer for Illumina Systems

TTAGGC

Index 7 Primer for Illumina Systems

CAGATC

Index 4 Primer for Illumina Systems

TGACCA


✔✔✔✔✔✔
✔✘✘✔✔✔

The following table lists some (but not all) valid index combinations that can be sequenced together:

Pool of 2 samples Index 6 and 12 Primers
Pool of 3 samples Index 4, 6, and 12 Primers
Pool of 6 samples Index 2, 4, 5, 6, 7, and 12 Primers

Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN) can generate 12 different, barcoded samples if each Index primer is used only once with Universal PCR Primer for Illumina Systems. Each Index Primer is supplied in sufficient amounts to generate two libraries, but these two libraries cannot be pooled together in one sequencing lane.

For 1-plex (no pooling), use any index primer with the universal PCR primer.


II. Index 1-12 Primers for Illumina Systems:

Each Index Primer for Illumina Systems is provided in volume of 10 µl.

Index 1 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAG-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

ATCACG

Index 2 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAG-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

CGATGT

Index 3 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATGCCTAAGTGACTGGA-
GTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TTAGGC

Index 4 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATTGGTCAGTGACTGGA-
GTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TGACCA

Index 5 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATCACTGTGTGACTGGA-
GTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ACAGTG

Index 6 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAG-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

GCCAAT

Index 7 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATGATCTGGTGACTGGAG-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

CAGATC

Index 8 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATTCAAGTGTGACTGGAG-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

ACTTGA

Index 9 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATCTGATCGTGACTGGAG-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

GATCAG

Index 10 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATAAGCTAGTGACTGGAG-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

TAGCTT

Index 11 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATGTAGCCGTGACTGGA-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

GGCTAC

Index 12 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAG-
TTCAGACGTGTGCTCTTCCGATC-s-T-3´

CTTGTA

Where -s- indicates phosphorothioate bond.


III. Set up the PCR Reaction

  1. Ensure that a valid combination of index primers is used. See Section I and II to verify that correct primer combinations have been selected.
  2. Add only one index primer () (5 µl) and 5 µl universal PCR primer () to each PCR tube. It is critical to change tips between tubes to avoid cross-contamination.
  3. Record the index primers added to each PCR tube.
  4. Add 25 µl Q5 PCR Master Mix () to each tube that contains primers.
  5. Add 15 µl of adaptor ligated ChIP DNA for a final volume of 50 µl to the corresponding tube. Gently pipette up and down 5–10 times to mix. It is critical to change tips between samples to avoid cross-contamination.
  6. Record the adaptor ligated DNA sample added to each PCR tube.
  7. Quickly centrifuge and perform PCR according to recommended cycling conditions (refer to the respective protocols in DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-DNA or CUT&RUN DNA starting samples).

APPENDIX: Quality Control of the Kit Components

The components in the SimpleChIP® ChIP-seq Multiplex Oligos for Illumina Systems (Single Index Primers) #29580 are individually validated by the functional testing listed below and must pass rigorous quality control standards. Furthermore, each set of components is functionally validated together by construction and sequencing of indexed libraries on the Illumina Systems sequencing platform.

I. Adaptor for Illumina Systems (15 μM) ()

5´-/5Phos/GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT C/ideoxyU/A CAC TCT TTC CCT ACA CGA CGC TCT TCC GAT C-s-T-3´

Quality Control Assays

  1. 16-Hour Incubation: 50 μl reactions containing this adaptor 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 a minimum of 5 μl of this adaptor with 1 μg of φX174 RF 1 DNA in assay buffer for 4 hours at 37°C in 50 μl reactions results in < 10% conversion to RF II as determined by agarose gel electrophoresis.
  3. Phosphatase Activity: Incubation of a minimum of 10 μl of this adaptor 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. RNase Activity: Incubation of this adaptor 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.

II. USER Enyzme ()

Supplied in: 50 mM KCl, 5 mM NaCl, 10 mM Tris-HCl (pH 7.4 @ 25°C), 0.1 mM EDTA, 1 mM DTT, 175 μg/ml BSA and 50% Glycerol

Quality Control Assays

  1. Non-Specific DNase Activity (16 Hour): A 50 μl reaction in NEBuffer 1 containing 1 μg of Lambda DNA and a minimum of 50 units of Uracil DNA Glycosylase incubated for 16 hours at 37°C results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. A 50 μl reaction in Endonuclease VIII Reaction Buffer containing 1 μg of Lambda-HindIII DNA and a minimum of 25 units of Endonuclease VIII incubated for 16 hours at 37°C results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
  2. Exonuclease Activity (Radioactivity Release): A 50 μl reaction in NEBuffer 1 containing 1 μg of a mixture of single and double-stranded [3H] E. coli DNA and a minimum of 50 units of Uracil DNA Glycosylase incubated for 4 hours at 37°C releases < 0.1% of the total radioactivity. A 50 μl reaction in Endonuclease VIII Reaction Buffer containing 1 μg of a mixture of single and double-stranded [3H] E. coli DNA and a minimum of 10 units of Endonuclease VIII incubated for 4 hours at 37°C releases < 0.5% of the total radioactivity.
  3. Endonuclease Activity (Nicking): A 50 μl reaction in UDG Reaction Buffer containing 1 μg of supercoiled φX174 DNA and a minimum of 50 units of Uracil DNA Glycosylase incubated for 4 hours at 37°C results in < 10% conversion to the nicked form as determined by agarose gel electrophoresis.
  4. Phosphatase Activity: Incubation of a minimum of 10 μl of USER Enzyme 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. Universal PCR Primer for Illumina Systems (10 μM) ()

5´-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC*T-3´

Quality Control Assays

  1. 16-Hour Incubation: 50 μl reactions containing this primer 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 primer and 1 μg T3 DNA incubated for 16 hours at 37°C also results in no detectable nonspecific nuclease degradation as determined by agarose gel electrophoresis.
  2. Endonuclease Activity: Incubation of a minimum of 5 μl of primer with 1 μg of φX174 RF I DNA in assay buffer for 4 hours at 37°C in 50 μl reactions results in < 10% conversion to RF II as determined by agarose gel electrophoresis.
  3. RNase Activity: Incubation of 1 μl of primer 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 a minimum of 10 μl of this primer 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.

IV. Index 1-12 Primers for Illumina Systems (10 μM) ()

Quality Control Assays

  1. 16-Hour Incubation: 50 μl reactions containing 1 μl Index [X] Primer for Illumina Systems 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 Index [X] Primer for Illumina Systems 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 Index [X] Primer for Illumina Systems 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 Index [X] Primer for Illumina Systems 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 Index [X] Primer for Illumina Systems in protein phosphatase assay buffer (1 M diethanolamine @ pH 9.8 and 0.5 mM MgCl2) containing 2.5 mM pnitrophenyl phosphate at 37°C for 4 hours yields no detectable p-nitrophenylene anion as determined by spectrophotometric analysis at 405 nm.

posted November 2017

revised January 2023

protocol id: 1626

Protocol Id: 1626

Multiplex Oligos for Illumina® (Dual Index Primers) (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. Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) contains adaptors and primers that are ideally suited for multiplex sample preparation for NG-seq on the Illumina® platform (Illumina, Inc.). This kit can be used to generate up to 96 distinct, barcoded ChIP-seq or CUT&RUN DNA libraries that can be combined into a single sequencing reaction.

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 provides enough reagents to support up to 96 DNA sequencing libraries, and must be used in combination with DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795.

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
ChIP-seq DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795
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. (red) Adaptor for Illumina® #42436
  2. (red) USER Enzyme #59713
  3. º (white) Index 501 Primer for Illumina® #86676
  4. º (white) Index 502 Primer for Illumina® #92596
  5. º (white) Index 503 Primer for Illumina® #29576
  6. º (white) Index 504 Primer for Illumina® #46325
  7. º (white) Index 505 Primer for Illumina® #67343
  8. º (white) Index 506 Primer for Illumina® #89663
  9. º (white) Index 507 Primer for Illumina® #27649
  10. º (white) Index 508 Primer for Illumina® #40566
  11. (orange) Index 701 Primer for Illumina® #60797
  12. (orange) Index 702 Primer for Illumina® #79999
  13. (orange) Index 703 Primer for Illumina® #18697
  14. (orange) Index 704 Primer for Illumina® #26125
  15. (orange) Index 705 Primer for Illumina® #39467
  16. (orange) Index 706 Primer for Illumina® #51808
  17. (orange) Index 707 Primer for Illumina® #58724
  18. (orange) Index 708 Primer for Illumina® #65787
  19. (orange) Index 709 Primer for Illumina® #75272
  20. (orange) Index 710 Primer for Illumina® #99422
  21. (orange) Index 711 Primer for Illumina® #10812
  22. (orange) Index 712 Primer for Illumina® #38569
  23. Index Primer Orange Tube Caps #54631
  24. Index Primer White Tube Caps #70942

Reagents Not Included:

  1. Enzymes and buffers appropriate for ChIP or CUT&RUN Illumina® library preparation: provided in DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795
  2. Nuclease-free Water #12931
  3. AMPure® XP Beads (Beckman Coulter, Inc. #A63881) or SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317)
  4. Freshly prepared 80% Ethanol
  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 Separation Rack #7017/#14654
  8. Bioanalyzer® and Agilent High Sensitivity DNA Kit (Agilent Technologies, Inc.) PCR tubes and PCR

Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) Protocol

I. Low Plexity Pooling Guidelines:

The dual index primer strategy utilizes two 8 base indices within each primer. Index 7 primers contain indices that are adjacent to the P7 sequence while index 5 primers contain indices that are adjacent to the P5 sequence. Dual indexing is enabled by adding a unique index to both ends of a sample to be sequenced. Up to 96 different samples can be uniquely indexed by combining each of the 12 index 7 primers with each of the 8 index 5 primers.

Illumina® NG-seq platforms use a red laser/LED to sequence A/C and a green laser/LED to sequence G/T. For each cycle, both the red and the green channel need to be read to ensure proper image registration (i.e. A or C must be present in each cycle, and G or T must be present in each cycle). If this color balance is not maintained, sequencing the index read could fail. Please check the sequences of each index to be used to ensure that you will have signal in both the red and green channels for every cycle. See example below:

ChIP Protocol Table 1

ChIP Protocol Table 2

The following table lists some (but not all) valid index combinations that can be sequenced together:

ChIP Protocol Table 3

Some other valid combinations are listed below. Choose a valid set of Index 7 primers and a valid set of Index 5 primers. Use each Index 7 primer with each i5 primer to form desired number of primer pairs for PCR amplification of desired number of libraries.

ChIP Protocol Table 4

II. Index 5 Primers for Illumina®:

Each Index 5 Primer for Illumina® is provided in volume of 60 µl.

ChIP Protocol Table 5

Where -s- indicates phosphorothioate bond.

III. Index 7 Primers for Illumina®:

Each Index 7 Primer for Illumina® is provided in a volume of 40 µl.

ChIP Protocol Table 6

Where -s- indicates phosphorothioate bond.

IV. Set up the PCR Reaction

  1. Ensure that a valid combination of index 7 and index 5 primers is used. See Section I and II to verify that correct primer combinations have been selected.
  2. Add only one Index 5 primer (º) (5 µl) and only one Index 7 primer () (5 µl) to each PCR tube. It is critical to change tips between tubes to avoid cross-contamination. If necessary, discard the original Index 5 white caps or Index 7 orange caps and apply new caps to avoid index cross-contamination.
  3. Record the Index 5 and Index 7 primers added to each PCR tube.
  4. Add 25 µl Q5® PCR Master Mix () to each tube that contains primers.
  5. Add 15 µl of adaptor ligated DNA for a final volume of 50 µl to the corresponding tube. Gently pipette up and down 5–10 times to mix. It is critical to change tips between samples to avoid cross-contamination.
  6. Record the adaptor ligated DNA sample added to each PCR tube.
  7. Quickly centrifuge and perform PCR according to recommended cycling conditions (refer to the respective protocols in DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795 for ChIP-DNA or CUT&RUN DNA starting samples).

APPENDIX: Quality Control of the Kit Components

The components in the Multiplex Oligos for Illumina® (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538 are individually validated by the functional testing listed below and must pass rigorous quality control standards. Furthermore, each set of components is functionally validated together by construction and sequencing of indexed libraries on the Illumina® sequencing platform.

I. Adaptor for Illumina® (15 µM) ()

5´-/5Phos/GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT C/ideoxyU/A CAC TCT TTC CCT ACA CGA CGC TCT TCC GAT C-s-T-3´

Quality Control Assays

  1. 16-Hour Incubation: 50 µl reactions containing this adaptor 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 a minimum of 5 µl of this adaptor with 1 µg of φX174 RF 1 DNA in assay buffer for 4 hours at 37°C in 50 µl reactions results in < 10% conversion to RF II as determined by agarose gel electrophoresis.
  3. Phosphatase Activity: Incubation of a minimum of 10 µl of this adaptor 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. RNase Activity: Incubation of this adaptor 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.

II. USER Enyzme ()

Supplied in: 50 mM KCl, 5 mM NaCl, 10 mM Tris-HCl (pH 7.4 @ 25°C), 0.1 mM EDTA, 1 mM DTT, 175 µg/ml BSA, and 50% Glycerol

Quality Control Assays

  1. Non-Specific DNase Activity (16 Hour): A 50 µl reaction in NEBuffer 1 containing 1 µg of Lambda DNA and a minimum of 50 units of Uracil DNA Glycosylase incubated for 16 hours at 37°C results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis. A 50 µl reaction in Endonuclease VIII Reaction Buffer containing 1 µg of Lambda-HindIII DNA and a minimum of 25 units of Endonuclease VIII incubated for 16 hours at 37°C results in a DNA pattern free of detectable nuclease degradation as determined by agarose gel electrophoresis.
  2. Exonuclease Activity (Radioactivity Release): A 50 µl reaction in NEBuffer 1 containing 1 µg of a mixture of single and double-stranded [3H] E. coli DNA and a minimum of 50 units of Uracil DNA Glycosylase incubated for 4 hours at 37°C releases < 0.1% of the total radioactivity. A 50 µl reaction in Endonuclease VIII Reaction Buffer containing 1 µg of a mixture of single and double-stranded [3H] E. coli DNA and a minimum of 10 units of Endonuclease VIII incubated for 4 hours at 37°C releases < 0.5% of the total radioactivity.
  3. Endonuclease Activity (Nicking): A 50 µl reaction in UDG Reaction Buffer containing 1 µg of supercoiled φX174 DNA and a minimum of 50 units of Uracil DNA Glycosylase incubated for 4 hours at 37°C results in < 10% conversion to the nicked form as determined by agarose gel electrophoresis.
  4. Phosphatase Activity: Incubation of a minimum of 10 µl of USER 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. Index 5 and Index 7 Primers for Illumina® (10 µM) (º)

Quality Control Assays

  1. 16-Hour Incubation: 50 µl reactions containing 1 µl Index [X] Primer for Illumina® 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 Index [X] Primer for Illumina® 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 Index [X] Primer for Illumina® 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 Index [X] Primer for Illumina® 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 Index [X] Primer for Illumina® 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.

posted November 2017

revised April 2022

protocol id: 1625

Protocol Id: 2806

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. Multiplex Oligos for Illumina Systems (Single Index Primers) (ChIP-seq, CUT&RUN) contains adaptors and primers that are ideally suited for multiplex sample preparation for NG-seq on the Illumina Systems platform. This kit can be used to generate up to 12 distinct, barcoded ChIP-seq or CUT&RUN DNA libraries that can be combined into a single sequencing reaction.

This product is compatible with SimpleChIP Enzymatic ChIP Kit (Magnetic Beads) #9003, SimpleChIP Plus Enzymatic ChIP Kit (Magnetic Beads) #9005, SimpleChIP Plus Sonication ChIP 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 DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 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. Libraries prepared from different starting amounts of DNA exhibit similiar Agilent Bioanalyzer System profiles, genome mapping rates, numbers of identified binding peaks, and signal-to-noise ratios across the whole genome.

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).

Limited Uses

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