Cat. # | Size | Price | Inventory |
---|---|---|---|
47415S | 1 Kit |
Product Includes | Volume (with Count) | Storage Temp | |||
---|---|---|---|---|---|
CUT&Tag Index 501 Primer for Illumina Systems | 1 x 30 µl | -20°C | |||
CUT&Tag Index 502 Primer for Illumina Systems | 1 x 30 µl | -20°C | |||
CUT&Tag Index 503 Primer for Illumina Systems | 1 x 30 µl | -20°C | |||
CUT&Tag Index 504 Primer for Illumina Systems | 1 x 30 µl | -20°C | |||
CUT&Tag Index 505 Primer for Illumina Systems | 1 x 30 µl | -20°C | |||
CUT&Tag Index 506 Primer for Illumina Systems | 1 x 30 µl | -20°C | |||
CUT&Tag Index 507 Primer for Illumina Systems | 1 x 30 µl | -20°C | |||
CUT&Tag Index 508 Primer for Illumina Systems | 1 x 30 µl | -20°C | |||
CUT&Tag Index 701 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 702 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 703 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 704 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 705 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 706 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 707 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 708 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 709 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 710 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 711 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag Index 712 Primer for Illumina Systems | 1 x 20 µl | -20°C | |||
CUT&Tag PCR Master Mix 63228 | 4 x 840 µl | -20°C |
Product Information
Next generation sequencing (NG-seq) is a high throughput method that can be used downstream of Cleavage Under Targets and Tagmentation (CUT&Tag) assay to identify and quantify target DNA enrichment across the entire genome. The CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems kit is 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 CUT&Tag DNA libraries that can be combined into a single sequencing reaction. This product is compatible with CUT&Tag DNA sample generated by the CUT&Tag pAG-Tn5 (Loaded) #79561 and DNA samples from other tagmentation assays, such as ATAC-seq. This product is not compatible with ChIP-DNA from SimpleChIP® Chromatin IP Kits (#9003, #9005, #56383) or the CUT&RUN DNA from CUT&RUN Assay Kit (#86652).
Compatible Reagent:
Non-Compatible Assay kits:
Required Reagents
Reagents Included:
Reagents Not Included:
SAFE STOP | This is a safe stopping point in the protocol, if stopping is necessary. |
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 systems 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:
GOOD | |||
---|---|---|---|
CUT&Tag Index 7 Primers for Illumina Systems | CUT&Tag Index 5 Primers for Illumina Systems | ||
Index 701 |
ATTACTCG |
Index 503 |
CCTATCCT |
Index 702 |
TCCGGAGA |
Index 504 |
GGCTCTGA |
Index 703 |
CGCTCATT |
Index 505 |
AGGCGAAG |
Index 704 |
GAGATTCC |
Index 506 |
TAATCTTA |
✔✔✔✔✔✔✔✔ | ✔✔✔✔✔✔✔✔ |
BAD | ||||
---|---|---|---|---|
CUT&Tag Index 7 Primers for Illumina Systems | CUT&Tag Index 5 Primers for Illumina Systems | |||
Index 701 |
ATTACTCG |
Index 502 |
ATAGAGGC |
|
Index 702 |
TCCGGAGA |
Index 504 |
GGCTCTGA |
|
Index 703 |
CGCTCATT |
Index 506 |
TAATCTTA |
|
Index 704 |
GAGATTCC |
Index 508 |
GTACTGAC |
|
✔✔✔✔✔✔✔✔ | ✔✔✘✔✔✘✔✘ |
The following table lists some (but not all) valid index combinations that can be sequenced together:
Plex | CUT&Tag Index 7 primers for Illumina Systems | CUT&Tag 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 CUT&Tag Index 7 primers and a valid set of CUT&Tag Index 5 primers. Use each CUT&Tag Index 7 primer with each CUT&Tag Index 5 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 |
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 |
Each CUT&Tag Index 5 Primer for Illumina systems is provided in a volume of 30 µl.
Product | Index Primer Sequence | Expected Index Primer Sequence Read |
CUT&Tag Index 501 Primer for Illumina Systems | 5´- AATGATACGGCGACCACCGAGATCTACACTA TAGCCTTCGTCGGCAGCGTCAGATGTG-s-T-3´ |
TATAGCCT |
CUT&Tag Index 502 Primer for Illumina Systems | 5´- AATGATACGGCGACCACCGAGATCTACACAT AGAGGCTCGTCGGCAGCGTCAGATGTG-s-T-3´ |
ATAGAGGC |
CUT&Tag Index 503 Primer for Illumina Systems | 5´- AATGATACGGCGACCACCGAGATCTACACCC TATCCTTCGTCGGCAGCGTCAGATGTG-s-T-3´ |
CCTATCCT |
CUT&Tag Index 504 Primer for Illumina Systems | 5´- AATGATACGGCGACCACCGAGATCTACACGG CTCTGATCGTCGGCAGCGTCAGATGTG-s-T-3´ |
GGCTCTGA |
CUT&Tag Index 505 Primer for Illumina Systems | 5´- AATGATACGGCGACCACCGAGATCTACACAG GCGAAGTCGTCGGCAGCGTCAGATGTG-s-T-3´ |
AGGCGAAG |
CUT&Tag Index 506 Primer for Illumina Systems | 5´- AATGATACGGCGACCACCGAGATCTACACTA ATCTTATCGTCGGCAGCGTCAGATGTG-s-T-3´ |
TAATCTTA |
CUT&Tag Index 507 Primer for Illumina Systems | 5´- AATGATACGGCGACCACCGAGATCTACACCA GGACGTTCGTCGGCAGCGTCAGATGTG-s-T-3´ |
CAGGACGT |
CUT&Tag Index 508 Primer for Illumina Systems | 5´- AATGATACGGCGACCACCGAGATCTACACGT ACTGACTCGTCGGCAGCGTCAGATGTG-s-T-3´ |
GTACTGAC |
Where -s- indicates phosphorothioate bond.
Each CUT&Tag Index 7 Primer for Illumina systems is provided in a volume of 20 µl.
Product | Index Primer Sequence | Expected Index Primer Sequence Read |
CUT&Tag Index 701 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATCGAGT AATGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
ATTACTCG |
CUT&Tag Index 702 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATTCTCC GGAGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
TCCGGAGA |
CUT&Tag Index 703 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATAATGA GCGGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
CGCTCATT |
CUT&Tag Index 704 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATGGAAT CTCGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
GAGATTCC |
CUT&Tag Index 705 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATTTCTG AATGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
ATTCAGAA |
CUT&Tag Index 706 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATACGAA TTCGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
GAATTCGT |
CUT&Tag Index 707 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATAGCTT CAGGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
CTGAAGCT |
CUT&Tag Index 708 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATGCGCA TTAGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
TAATGCGC |
CUT&Tag Index 709 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATCATAG CCGGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
CGGCTATG |
CUT&Tag Index 710 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATTTCGC GGAGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
TCCGCGAA |
CUT&Tag Index 711 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATGCGCG AGAGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
TCTCGCGC |
CUT&Tag Index 712 Primer for Illumina Systems | 5´- CAAGCAGAAGACGGCATACGAGATCTATC GCTGTCTCGTGGGCTCGGAGATGTG-s-T-3´ |
AGCGATAG |
Where -s- indicates phosphorothioate bond.
Before starting:
Reagents | Volume for 1 PCR Reaction (70 µl) |
---|---|
CUT&Tag DNA (or any tagmentated DNA) | 30 µl |
CUT&Tag PCR Master Mix | 35 µl |
CUT&Tag Index 7 Primer for Illumina Systems (10 µM) | 2.5 µl |
CUT&Tag Index 5 Primer for Illumina Systems (10 µM) | 2.5 µl |
NOTE: It is critical to change tips between tubes to avoid cross-contamination. If starting with less than 30 µl of CUT&Tag DNA, add DNAse-free water to bring the volume up to 30 µl.
NOTE: It is critical to change tips between samples to avoid cross-contamination.
a. | Gap Filling | 58°C for 5 min |
b. | Gap Filling Extension | 72°C for 5 min |
c. | Initial Denaturation | 98°C for 30 sec |
d. | Denaturation | 98°C for 10 sec |
e. | Anneal and Extension | 60°C for 11 sec |
|
For between 20,000 and 100,000 cells per CUT&Tag reaction, repeat steps d and e for a total of 13 cycles. | |
|
For 20,000 and less cells per CUT&Tag reaction, repeat steps d and e for a total of 14-16 cycles. | |
|
Note: excessive PCR cycles lead to lower library diversity and/or higher duplication rate of NGS reads. | |
f. | Final Extension | 72°C for 1 min |
g. | Hold | 4°C |
Before starting:
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.
NOTE: While CUT&Tag DNA libraries generated for histone modifications typically show robust signal in Bioanalyzer or TapeStation systems analysis, libraries generated for non-histone proteins such as transcription factors and cofactors often have very weak or even no visible signal using Bioanalyzer or TapeStation systems, but still generate NG-sequencing results with high mapping rates, high numbers of identified binding peaks, and acceptable signal-to-noise ratios across the whole genome. Therefore, we recommend sequencing DNA library preps from transcription factor and cofactor CUT&Tag reactions that do not show a signal in Bioanalyzer or TapeStation systems analysis.
Protocol Id: 2827
All Species Expected
Similar to Cleavage Under Targets and Release Using Nuclease (CUT&RUN), Cleavage Under Targets and Tagmentation (CUT&Tag) is a powerful technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1-3). CUT&Tag has many of the same advantages as the CUT&RUN assay in that it provides a rapid, robust, and true low cell number protocol for detection of protein-DNA interactions in the cell. In addition, the CUT&Tag assay adds an in situ adaptor DNA ligation step carried out by the pAG-Tn5 enzyme, in which an adaptor DNA is ligated directly to antibody-targeted chromatin DNA fragments in the cell. As a result, subsequent DNA library preparation is much faster and easier than library preparation following the CUT&RUN assay, free from DNA end repair, A-tailing, and adaptor ligation in vitro. CUT&Tag works very well for analyzing histone modifications, in addition to mapping some transcription factor and cofactor binding.
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