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Multiplex Oligos for Illumina Systems (Dual Index Primers) (ChIP-seq, CUT&RUN)
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Multiplex Oligos for Illumina Systems (Dual Index Primers) (ChIP-seq, CUT&RUN) #47538

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Cat. # Size Qty. Price
47538S		 1 Kit  (96 assays)

Custom Formulation

Product Includes Volume (with Count) Storage Temp
Adaptor for Illumina Systems 1 x 960 µl -20°C
USER Enzyme 1 x 288 µl -20°C
Index Primer Orange Tube Caps 1 x 24 ea RT
Index Primer White Tube Caps 1 x 16 ea RT
Index 501 Primer for Illumina Systems 1 x 60 µl -20°C
Index 502 Primer for Illumina Systems 1 x 60 µl -20°C
Index 503 Primer for Illumina Systems 1 x 60 µl -20°C
Index 504 Primer for Illumina Systems 1 x 60 µl -20°C
Index 505 Primer for Illumina Systems 1 x 60 µl -20°C
Index 506 Primer for Illumina Systems 1 x 60 µl -20°C
Index 507 Primer for Illumina Systems 1 x 60 µl -20°C
Index 508 Primer for Illumina Systems 1 x 60 µl -20°C
Index 701 Primer for Illumina Systems 1 x 40 µl -20°C
Index 702 Primer for Illumina Systems 1 x 40 µl -20°C
Index 703 Primer for Illumina Systems 1 x 40 µl -20°C
Index 704 Primer for Illumina Systems 1 x 40 µl -20°C
Index 705 Primer for Illumina Systems 1 x 40 µl -20°C
Index 706 Primer for Illumina Systems 1 x 40 µl -20°C
Index 707 Primer for Illumina Systems 1 x 40 µl -20°C
Index 708 Primer for Illumina Systems 1 x 40 µl -20°C
Index 709 Primer for Illumina Systems 1 x 40 µl -20°C
Index 710 Primer for Illumina Systems 1 x 40 µl -20°C
Index 711 Primer for Illumina Systems 1 x 40 µl -20°C
Index 712 Primer for Illumina Systems 1 x 40 µl -20°C

Storage

Upon receipt, #54631 and #70942 tube caps should be removed from the kit box and stored at room temperature. Store remaining components at -20ºC. This product is stable for 12 months if stored properly.

Protocol

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Multiplex Oligos for Illumina Systems (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 Systems (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 Systems 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 Systems 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 Systems (ChIP-seq, CUT&RUN) #56795.

Compatible Assay kits:
CUT&RUN Assay Kit #86652
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 Systems (ChIP-seq, CUT&RUN) #56795

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. º (white) Index 501 Primer for Illumina Systems #86676
  4. º (white) Index 502 Primer for Illumina Systems #92596
  5. º (white) Index 503 Primer for Illumina Systems #29576
  6. º (white) Index 504 Primer for Illumina Systems #46325
  7. º (white) Index 505 Primer for Illumina Systems #67343
  8. º (white) Index 506 Primer for Illumina Systems #89663
  9. º (white) Index 507 Primer for Illumina Systems #27649
  10. º (white) Index 508 Primer for Illumina Systems #40566
  11. • (orange) Index 701 Primer for Illumina Systems #60797
  12. • (orange) Index 702 Primer for Illumina Systems #79999
  13. • (orange) Index 703 Primer for Illumina Systems #18697
  14. • (orange) Index 704 Primer for Illumina Systems #26125
  15. • (orange) Index 705 Primer for Illumina Systems #39467
  16. • (orange) Index 706 Primer for Illumina Systems #51808
  17. • (orange) Index 707 Primer for Illumina Systems #58724
  18. • (orange) Index 708 Primer for Illumina Systems #65787
  19. • (orange) Index 709 Primer for Illumina Systems #75272
  20. • (orange) Index 710 Primer for Illumina Systems #99422
  21. • (orange) Index 711 Primer for Illumina Systems #10812
  22. • (orange) Index 712 Primer for Illumina Systems #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 Systems 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
  8. Agilent Bioanalyzer Systems and Agilent High Sensitivity DNA Kit (Agilent Technologies, Inc.)
  9. PCR tubes and PCR machine

Multiplex Oligos for Illumina Systems (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 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 701 Primer for Illumina Systems

ATTACTCG

 Index 503 Primer for Illumina Systems

CCTATCCT
Index 702 Primer for Illumina Systems

TCCGGAGA

 Index 504 Primer for Illumina Systems

GGCTCTGA
Index 703 Primer for Illumina Systems

CGCTCATT

 Index 505 Primer for Illumina Systems

AGGCGAAG
Index 704 Primer for Illumina Systems

GAGATTCC

 Index 506 Primer for Illumina Systems

TAATCTTA

✔✔✔✔✔✔✔✔
✔✔✔✔✔✔✔✔
Index 7 Primers for Illumina Systems Index 5 Primers for Illumina Systems
Index 701 Primer for Illumina Systems

ATTACTCG

 Index 502 Primer for Illumina Systems

ATAGAGGC
Index 702 Primer for Illumina Systems

TCCGGAGA

 Index 504 Primer for Illumina Systems

GGCTCTGA
Index 703 Primer for Illumina Systems

CGCTCATT

 Index 506 Primer for Illumina Systems

TAATCTTA
Index 704 Primer for Illumina Systems

GAGATTCC

 Index 508 Primer for Illumina Systems

GTACTGAC

✔✔✔✔✔✔✔✔
✔✔✘✔✔✘✔✘

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

Plex Index 7 primers for Illumina Systems Index 5 Primers for Illumina Systems
2 Index 701 and Index 702
Index 703 and Index 704
Index 705 and Index 706
Index 707 and Index 708
Index 709 and Index 710
Index 711 and Index 712 		Any Index 5 Primer
3 Index 701, Index 702 and Index 703
Index 703, Index 704 and Index 705
Index 705, Index 706 and Index 707
Index 707, Index 708 and Index 709
Index 709, Index 710 and Index 711 		Any Index 5 Primer
4 Index 701, Index 702, Index 703 and Index 704
Index 703, Index 704, Index 705 and Index 706
Index 705, Index 706, Index 707 and Index 708
Index 707, Index 708, Index 709 and Index 710
Index 709, Index 710, Index 711 and Index 712 		Any Index 5 Primer
5-12 Any valid Index 7 4-plex combination with any other i7 Primers (as needed) Any Index 5 Primer
> 12
 Any valid Index 7 4-plex combination with any other i7 primer (as needed) Index 501, Index 502 and any other Index 5 primer (as needed)
Index 503, Index 504 and any other Index 5 primer (as needed)
Index 505, Index 506 and any other Index 5 primer (as needed)
Index 507, Index 508 and any other Index 5 primer (as needed)

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.

Pool of 12 samples (1) A set of 4 Index 7 primers * A set of 3 Index 5 primers
(2) A set of 3 Index 7 primers * A set of 4 Index 5 primers
(3) A set of 6 Index 7 primers * A set of 2 Index 5 primers
(4) A set of 12 Index 7 primers * A set of 1 Index 5 primers
Pool of 26 samples (1) A set of 6 Index 7 primers * A set of 4 Index 5 primers
Plus any of the Index 7 primers with any other two Index 5 primers (besides the set of 4)
(2) A set of 6 Index 7 primers * A set of 5 Index 5 primers
Use 26 of the 30 primer pairs to amplify 26 libraries

II. Index 5 Primers for Illumina Systems:

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

Index 501 Primer for Illumina Systems 5´-AATGATACGGCGACCACCGAGATCTACACTATAGCCTA-
CACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´

TATAGCCT
Index 502 Primer for Illumina Systems 5´-AATGATACGGCGACCACCGAGATCTACACATAGAGGCA-
CACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´

ATAGAGGC
Index 503 Primer for Illumina Systems 5´-AATGATACGGCGACCACCGAGATCTACACCCTATCCTA-
CACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´

CCTATCCT
Index 504 Primer for Illumina Systems 5´-AATGATACGGCGACCACCGAGATCTACACGGCTCTGAA-
CACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´

GGCTCTGA
Index 505 Primer for Illumina Systems 5´-AATGATACGGCGACCACCGAGATCTACACAGGCGAAGA-
CACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´

AGGCGAAG
Index 506 Primer for Illumina Systems 5´-AATGATACGGCGACCACCGAGATCTACACTAATCTTAA-
CACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´

TAATCTTA
Index 507 Primer for Illumina Systems 5´-AATGATACGGCGACCACCGAGATCTACACCAGGACGTA-
CACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´

CAGGACGT
Index 508 Primer for Illumina Systems 5´-AATGATACGGCGACCACCGAGATCTACACGTACTGACA-
CACTCTTTCCCTACACGACGCTCTTCCGATC-s-T-3´

GTACTGAC

Where -s- indicates phosphorothioate bond.

III. Index 7 Primers for Illumina Systems:

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

Index 701 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ATTACTCG
Index 702 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATTCTCCGGAGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TCCGGAGA
Index 703 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATAATGAGCGGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

CGCTCATT
Index 704 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATGGAATCTCGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GAGATTCC
Index 705 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATTTCTGAATGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

ATTCAGAA
Index 706 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATACGAATTCGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

GAATTCGT
Index 707 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATAGCTTCAGGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

CTGAAGCT
Index 708 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATGCGCATTAGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TAATGCGC
Index 709 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATCATAGCCGGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

CGGCTATG
Index 710 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATTTCGCGGAGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TCCGCGAA
Index 711 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATGCGCGAGAGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

TCTCGCGC
Index 712 Primer for Illumina Systems 5´-CAAGCAGAAGACGGCATACGAGATCTATCGCTGTGACTG-
GAGTTCAGACGTGTGCTCTTCCGATC-s-T-3´

AGCGATAG

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 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 Multiplex Oligos for Illumina Systems (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 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 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 Systems (10 µM) (º•)

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 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 March 2023

protocol id: 1625

Protocol Id: 1625

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: 2805

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 (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 Systems platform. 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. 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 Systems (ChIP-seq, CUT&RUN) #56795.

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 similar Agilent Bioanalyzer System profiles, genome mapping rates, numbers of identified binding peak, 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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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.

For Research Use Only. Not for Use in Diagnostic Procedures.
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