Multiplex Oligos for Illumina Protocol (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

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.