TAM-ChIP anti-rabbit conjugate
Catalog No: 53126
Lot No: 23517002
Format: 16 rxns
Backgound: Active Motif’s TAM-ChIP combines antibody directed genomic targeting and NGS library preparation in one
step. First, a ChIP-validated antibody is used to target a genomic region of interest, such as a histone or transcription
factor binding site. Then, a TAM-ChIP anti-species antibody that is conjugated to barcoded sequencing adapters andTsTn5 transposase is added to each reaction. Activation of the transposase cuts the nearby DNA and pastes the antibodyassociated adapters into the DNA sequence. Following immunoprecipitation, enriched DNA is ready for library®

amplification and sequencing using Illumina platforms.
ContentsStorage• Anti-rabbit conjugate• Oligonucleotide• pMENTS AR• TsTn5 transposase• TAM-ChIP Index 1-16
• Amplification primer mix• 5X Tagmentation Buffer

4°C
-20°C
-20°C
-20°C
-20°C
-20°C
-20°C

Concentrations forLot 23517002
1.2 mg/ml
100 µM
100 µM
142.4 µM
0.5 µM
10 µM ea
5X

Volumes to assemble TAMChIP antibody conjugate
3.3 µlN/A
0.76 µl0.53 µl

Volumes for
Oligo assembly
N/A
2 µl
2 µl
1.4 µlRelated Products (not included)• TAM-ChIP Assay Reagents (Catalog No. 53128)

TAM-ChIP illustration.
Intact cells are fixed with formaldehyde to preserve
protein/DNA interactions. DNA is then sheared using
sonication and incubated with a ChIP-validated
antibody directed against the protein of interest. ATAM-ChIP antibody conjugate containing Illuminacompatible sequencing adapters and transposase isadded. Activation of the transposase cuts the DNA
surrounding the genomic region of interest andpastes the barcoded adapters into the DNAsequences. The protein/DNA complexes areimmunoprecipitated, DNA cross-links are reversedand proteins are degraded. ChIP enriched DNA isready for library amplification and sequencing.

Quality Control: The TAM-ChIP anti-rabbit conjugate is tested in combination with Active Motif’s TAM-ChIP Assay
Reagents (Catalog No. 53128) using 10 µg of MCF-7 chromatin in combination with 4 µg CTCF antibody and 4 µg ofassembled TAM-ChIP anti-rabbit conjugate according to the instructions provided below. Enriched DNA is PCRamplified and size selected. ChIP-Seq is performed using the Illumina NextSeq 500 and sequencing results are mapped
to the reference genome.

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Protocol Guidelines: The following guidelines are provided for the use of the TAM-ChIP anti-mouse conjugate. Use only
the values supplied on the appropriate lot-specific data sheet. If using in conjunction with TAM-ChIP assay reagent (Cat.
No. 53128), please follow that manual and the lot-specific information on page 1.
Sample Preparation:
1. Prepare chromatin from cultured cell lines or tissue samples using your preferred protocol or Active Motif’s TAM-ChIP
Assay Reagents (Catalog No. 53128). We suggest formaldehyde fixation (1% final concentration) for 15 minutes at room
temperature followed by addition of a glycine stop solution for 5 minutes at room temperature.
Note: Due to the sensitivity of the TsTn5 transposase to SDS, it is important that buffers used during cell lysis andchromatin sonication do not contain SDS concentrations that exceed 0.1% SDS. The maximum amount ofSDS that can be used in the immunoprecipitation reaction should not exceed 0.02% final concentration.
2. Sonicate chromatin to achieve a sheared size of 200-1000 bp. To prevent overheating and denaturation of chromatin,samples should be kept on ice as much as possible during shearing, and shearing should be performed discontinuously(e.g. sonicate for 20 seconds, then place on ice for 30 seconds before sonicating again for 20 seconds, etc.)
3. Remove a 25 µl sample of sonicated chromatin for chromatin quantification. Reverse the protein cross-links andProteinase K treat the chromatin sample. Read the absorbance on a NanoDrop or other spectrophotometer at 260nm todetermine the DNA concentration. Use a Bioanalyzer or agarose gel to confirm the size of the sheared chromatin.
Recommendations for use with ChIP protocols other than TAM-ChIP:
1. We suggest performing the immunoprecipitation reaction using 5 - 10 µg chromatin and 4 µg ChIP-validated antibody
per reaction. We also recommend setting up a ChIP reaction without primary antibody to serve as a tagmentation
control. This control will show the effects of the transposase in the absence of genomic targeting and provide abackground reference during sequencing. Incubate ChIP reactions on an end-to-end rotator at 4°C overnight.
2. The next day, prepare the TAM-ChIP antibody conjugate and the Oligo assembly using the lot-specific information
provided on page 1 of the lot-specific data sheet. We recommend using an equivalent mass for the TAM-ChIP conjugate
as the ChIP-Seq antibody (4 µg). Use separate tubes to prepare the antibody conjugate and the oligo assembly. Thevolumes provided are for 1 reaction, please scale up accordingly.
Antibody Conjugate

Oligo Assembly

Reagent
TAM-ChIP Antibody conjugate
pMENTS AM (100 µM)
Incubate for 30 minutes at RT

1 reaction
See page 1
See page 1

Reagent
Oligonucleotide (100 µM)
pMENTS AM (100 µM)
Incubate for 30 minutes at RT

1 reaction
See page 1
See page 1

TsTn5 transposase
Incubate for 1 hour at RT

See page 1

TsTn5 transposase
See page 1
Incubate for 1 hour at RT (Keep at RT until ready to use)

3. Add the 4 µg of the assembled TAM-ChIP anti-mouse conjugate to each ChIP reaction from Step 1. Incubate on anend-to-end rotator at 4°C for 1 hour.
4. Adjust the amount of DTT in your reaction to a final concentration of 0.35 mM and incubate an end-to-end rotator at37°C for 1 hour.
5. Capture your ChIP reactions using Protein G beads. Wash to remove unbound DNA. The inclusion of 0.1% SDS in
the wash buffer will improve your wash efficiency. Resuspend your ChIP reactions in 400 µl buffer without SDS.
6. Activate the TsTn5 transposase with the addition of 5X Tagmentation Buffer. Add 100 µl Tagmentation buffer to400 µl ChIP reaction for a final volume of 500 µl.
7. Adjust the amount of DTT in your reaction to a final concentration of 0.35 mM (1.75 µl of 10 mM DTT stock).
8. Incubate on an end-to-end rotator at 37°C for 1 hour.
9. Wash beads and elute the chromatin.
10. Clean up the eluted DNA using phenol/chloroform and phase lock tubes followed by either ethanol precipitation orcolumn purification. Elute in 25 µl 10 mM Tris-Cl, pH 8.5.
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Library Amplification:
1. PCR amplify the ChIP enriched DNA. Below is an example PCR reaction. We strongly recommend using Q5 HighFidelity polymerase (New England Biolabs, M0491) to ensure good amplification efficiency. Other polymerase mixtures
may require optimization for efficient amplification. Follow the recommendations of your manufacturer’s PCR reagents.
Reagent
1 reaction
ChIP DNA
20 µl
TAM-Index primer* (0.5 µM)
5 µl
Amplification primer mix (10 µM each)
5 µl
5X Q5 Reaction Buffer
10 µl
Q5 High-Fidelity Polymerase
0.5 µldNTPs (10 mM)
1 µl
Sterile H2O
8.5 µlTotal volume50 µl*If combining multiple TAM-ChIP samples together for sequencing, make sure to use unique TAM-ChIP Index
primers for each sample.
Thermal Cycler Program:
98°C for 30 seconds
63°C for 3 minutes
98°C for 10 seconds
63°C for 30 seconds
72°C for 30 seconds

x14 cycles

4°C Hold
Proceed immediately to Library Size Selection
2. Size select the ChIP libraries using AMPure beads and 70% ethanol to 200-500 bases.
3. Assess chromatin size using TapeStation or equivalent method.
4. Sequence using an Illumina platform.

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For sequencing, we recommend 30 million reads. If combining multiple TAM-ChIP samples together for sequencing,make sure to use unique TAM-ChIP Index primers for each sample. The TAM-ChIP Index (i7) contains an 8 bp random
molecular identified (MID) followed by a 3 bp sample barcode for a total of 11 bp. The sequences below represent thetrue sequence and the NextSeq 500 sequence of index (i7). (If using different instruments determine if the truesequence or reverse complement should be entered).
True Sequence
TAM-ChIP Index 1 NNNNNNNNTCG
TAM-ChIP Index 2 NNNNNNNNGAC
TAM-ChIP Index 3 NNNNNNNNACG
TAM-ChIP Index 4 NNNNNNNNGCT
TAM-ChIP Index 5 NNNNNNNNTGA
TAM-ChIP Index 6 NNNNNNNNCGT
TAM-ChIP Index 7 NNNNNNNNCTA
TAM-ChIP Index 8 NNNNNNNNGTA
TAM-ChIP Index 9 NNNNNNNNAGT
TAM-ChIP Index 10 NNNNNNNNATC
TAM-ChIP Index 11 NNNNNNNNTAG
TAM-ChIP Index 12 NNNNNNNNCAG
TAM-ChIP Index 13 NNNNNNNNTAC
TAM-ChIP Index 14 NNNNNNNNGTCTAM-ChIP Index 15 NNNNNNNNCAC
TAM-ChIP Index 16 NNNNNNNNACA

NextSeq 500 Sequence
NNNNNNNNCGA
NNNNNNNNGTC
NNNNNNNNCGT
NNNNNNNNAGC
NNNNNNNNTCA
NNNNNNNNACG
NNNNNNNNTAG
NNNNNNNNTAC
NNNNNNNNACT
NNNNNNNNGAT
NNNNNNNNCTA
NNNNNNNNCTG
NNNNNNNNGTA
NNNNNNNNGAC
NNNNNNNNGTG
NNNNNNNNTGT

The antibody index (i5) is an 8 bp sequence of which 3 bp are used for analysis. The true sequence for the anti-mouse
conjugate i5 index is ATA and the NextSeq 500 sequence is TAT.
We recommend running the machine in standalone mode to allow use of different length sequencing of the i5 and i7indexes (11 on i7 and 8 on i5). If using BaseSpace, you will need the same number of reads on each end (11 and 11).
The type of sequencing to run may depend on your instrument, cartridge capacity and application. We routinely perform
singe-end sequencing, 73 cycles.
For additional information regarding data analysis and de-duplication of the MIDs, please visit Active Motif’s website
www.activemotif.com/tamchip and download the Guidelines for TAM-ChIP Molecular Identifier (MID) Analysis from the“Documents” tab. Or contact Technical Support at tech_service@activemotif.com

Guarantee: This product is for research use only and is not for use in diagnostic procedures. This product is guaranteedfor 6 months from date of arrival.

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