TITLE: NanoTag
AUTHORS: Maria Dimitriu, Leonard Steg, Rodrigo Arzate-Mejia, Isabelle Mansuy

[DESCRIPTION]
Background: Genome-wide profiling of DNA-protein interactions in cells can provide important information about mechanisms of gene regulation. Most current methods for genome-wide profiling of DNA-bound proteins such as ChIP-seq and CUT&Tag use conventional IgG antibodies to bind the target protein(s). This limits their applicability to targets with available high affinity and specificity antibodies and prevents their use for other targets. Here we describe NanoTag, an IgG-free method derived from CUT&Tag to profile DNA-protein interactions. NanoTag is based on a fusion between an anti-GFP nanobody and Tn5 transposase that can map GFP-tagged proteins associated with chromatin in a fast, cost-effective and animal-free manner.
Results: We used NanoTag to indirectly profile the histone mark H3K4me3 genome-wide via its binding partner TATA box-binding protein-associated factor 3 (TAF3) and the transcription factors Nanog and CTCF in mouse embryonic stem cells (mESCs). NanoTag results show high inter-replicate reproducibility, high signal-to-noise ratio and strong correlation with CUT&Tag datasets, validating its accuracy and reliability.
Conclusions: NanoTag provides a novel, flexible and cost-effective IgG-free method to generate high resolution DNA-binding profiles in cells and tissues. 
 

[STEPS]
SECTION: Cell collection
1. Remove media from culture dish and wash with D-PBS.
SECTION: Cell collection
2. Dissociate cells using Accutase and count cells. 

 

2. 
SECTION: Cell collection
3. Wash cells with 1 mL Wash buffer and resuspend cells at 1 million cells/mL of Wash buffer. 400 µL per sample. Transfer in a 2 mL tube. Use 400,000 cells per NanoTag sample. 

2. 
SECTION: Binding cells to beads
5. Prepare concanavalin A beads:

SECTION: Binding cells to beads
5.1. Withdraw 10 uL of bead slurry per sample (i.e. use 170 µL for 16 sample), transfer it into 1.6 mL Binding buffer and mix by pipetting. 

SECTION: Binding cells to beads
5.2. Place tube on magnet until solution is clear. Remove the liquid, then remove tube from magnet and resuspend in 1.5 mL Binding buffer. Mix by pipetting.

SECTION: Binding cells to beads
5.3. Place tube on magnet until solution is clear, then remove the liquid and resuspend the slurry in the original volume (170 µL for 16 samples) of Binding buffer. Keep the beads at room temperature until cells/nuclei are ready.

SECTION: Binding cells to beads
6. Add the correct amount of beads to the corresponding amount of cell suspension (10 µL of bead slurry and 400 µL of cell suspension) per sample while gently vortexing. 

SECTION: Binding cells to beads
7. Mix by pipetting and incubate on an end-over-end rotator for 15 min . During the incubation, continue with step 8. 

2. 
SECTION: Tagmentation
8. During incubation of step 6 mix the correct amount of GFP nanobody-Tn5 transposomes (2 µL per sample for 23 micromolar (µM) protein) and Dig-wash buffer (100 µL per sample). 

SECTION: Tagmentation
9. After the incubation from step 6 aliquot the bead-cell suspension to the corresponding amount of tubes. 
SECTION: Tagmentation
10. Place tubes on the magnet until solution is clear. Remove the liquid, remove tubes from magnet and resuspend beads in 100 µL of cold GFP nanobody-Tn5-containing Dig-wash buffer by gently pipetting. 

SECTION: Tagmentation
11. Incubate on a nutator   at 4 °C  to allow the GFP nanobody-Tn5 to bind to its target. 

SECTION: Tagmentation
12. The next day place tubes on the magnet until solution is clear and remove the liquid. Wash beads with 1 mL of Dig-med buffer. Incubate on nutator for 5 min . 

SECTION: Tagmentation
13. Repeat step 11 once to remove any anti-GFP nanobody-Tn5 protein that is unspecifically bound. 
SECTION: Tagmentation
14. Resuspend beads in 300 µL of Tagmentation buffer and incubate at 37 °C for 60 min . 

2. 
SECTION: Tagmentation stop and DNA extraction
15. Add 10 µL 0.5M EDTA, 3 µL 10% SDS and 2.5 µL of 20 mg/mL Proteinase K to each sample. Vortex tubes on full speed for 2-3 seconds. 

SECTION: Tagmentation stop and DNA extraction
16. Incubate at 55 °C for 60 min . 

SECTION: Tagmentation stop and DNA extraction
17. Add 300 µL Phenol:Chloroform:Isoamyl Alcohol and full speed vortex for 2s. 

SECTION: Tagmentation stop and DNA extraction
18. Transfer solution to a phase-lock tube and centrifuge 16000 x g, 3 min .

SECTION: Tagmentation stop and DNA extraction
19. Transfer the aqueous (top) layer to a fresh 1.5 mL tube containinng 750 µL 100% ethanol, pipet up and down to mix and keep tubes on ice . 

SECTION: Tagmentation stop and DNA extraction
20. Centrifuge 16000 x g, 10 min, 4 °C . Pour off the liquid and quickly drain on a paper towel.

SECTION: Tagmentation stop and DNA extraction
21. Add 1 mL 100% ethanol and centrifuge 16000 x g, 1 min, 4 °C . Pour off the liquid and let tubes dry on a paper towel for about 10 minutes.

SECTION: Tagmentation stop and DNA extraction
22. When tubes are dry add 25 µL Elution buffer and full-speed vortex to dissolve the DNA. 

SECTION: Tagmentation stop and DNA extraction
23. Store tagmented DNA at -20 °C .

2. 
SECTION: Library preparation
24. To identify the optimal amount of cycles to perform during the library amplification, perform a qPCR:
SECTION: Library preparation
24.1. Create a master mix by adding 2 µL of i5 primer and 2 µL of i7 primer, 0.15 µL 100X SYBR Green dye,  0.15 µL water and  0.85 µL NEBNext PCR Master mix per sample. 
SECTION: Library preparation
24.2. Add 12.5 µL of master mix to each well of a qPCR plate. Add 2.5 µL of tagmented DNA to each well and run the qPCR using the following program:

Cycle 1:  72 °C for 5 min (gap filling)
Cycle 2: 98 °C for 30 sec
Cycle 3: 98 °C for 10 sec
Cycle 4: 63 °C for 30 sec
Cycle 5: 72 °C for 60 sec
Repeat Cycles 3-4-5 20 times
SECTION: Library preparation
24.3. Check the amount of cycles necessary to achieve 1/3 of the maximum fluorescence for each sample and perform library amplification using that amount of cycles. 
 
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SECTION: Library preparation
25. Once the amount of cycles has been determined according to step 23 amplify libraries:
SECTION: Library preparation
25.1. Transfer 21 µL of tagmented DNA from each sample into a PCR tube. Add 2 µL of i5 index,  2 µL of i7 index (use different i5 and i7 combinations for each library) and 25 µL of NEBNext PCR master mix, mix and briefly spin down the sample then perform the following PCR program:

Cycle 1:  72 C for 5 min (gap filling)
Cycle 2: 98 °C for 30 sec
Cycle 3: 98 °C for 10 sec
Cycle 4: 63 °C for 10 sec
Repeat Cycles 3-4 for the amount of times determined in step 23
72°C for 1 min and hold at 8 °C

SECTION: Library preparation
26. Add 60 µL AMPure beads to each library. Pipet up and down 10x to mix. Briefly spin down and incubate for 10 min. Place on magnet and allow solution to clear. 

SECTION: Library preparation
27. Remove the liquid and wash beads 2x with 200 µL of 80% ethanol with the tubes still on the magnet. After the second wash remove the liquid using 2 pipetting steps.

SECTION: Library preparation
28. Add 20 µL Tris-HCl pH 8.0 and full-speed vortex. Incubate for 5 min. Pleace on magnet and allow solution to clear. Transfer liquid to fresh tubes. 

SECTION: Library preparation
29. Check the library quality using a fragment analyzer (e.g. Tapestation).
 

2. 
SECTION: Sequencing
30. NanoTag libraries can be sequenced using Illumina platforms. Before sequencing, serially dilute the libraries to equal molarity and pool to the desired molarity according to the sequencing platform specifications. We recommend adding 10% PhiX spike-in to the pool and performing paired-end sequencing using 50bp reads. 
SECTION: Cell collection
4. Take 400 µL of cell suspension (equivalent to 400k cells) per sample. 

2. 
2. 
2. 
SECTION: Preparing GFP nanobody-Tn5 transposomes
31. Transform LysY/lq competent E. coli with pMD1 plasmid encoding GFP nanobody-Tn5 with a intein-CBD tag. 

SECTION: Preparing GFP nanobody-Tn5 transposomes
32. Inoculate 2L of LB (+100ug/mL Ampicilin) with 1 colony and induce overexpression of GFP nanobody-Tn5 with 0.25 mM IPTG overnight at 18 °C . 

SECTION: Preparing GFP nanobody-Tn5 transposomes
33. The next day collect the bacterial medium, centrifuge and freeze the pellet. 
SECTION: Preparing GFP nanobody-Tn5 transposomes
34. Resuspend the pellet in 40 mL HEGX buffer and sonicate for 2.5 min using 10s cycles at 70% amplitude. 
SECTION: Preparing GFP nanobody-Tn5 transposomes
35. Centrifuge for 30 min at 20,000xg.
SECTION: Preparing GFP nanobody-Tn5 transposomes
36. Collect the supernatant and add 1.1 mL of 10% PEI (pH 7.5) to 40 mL of supernatant. 
SECTION: Preparing GFP nanobody-Tn5 transposomes
37. Centrifuge for 20 min at 20,000xg.
SECTION: Preparing GFP nanobody-Tn5 transposomes
38. Bind the supernatant tp chitin beads for 2h at 4 °C  in a disposable column. Wash thoroughly with 10 column volumes of HEGX buffer. 

SECTION: Preparing GFP nanobody-Tn5 transposomes
39. Induce cleavage of the GFP nanobody-Tn5 from its intein tag by incubating the column overnight at 4 °C  in HEGX buffer + 100 mM DTT. 

SECTION: Preparing GFP nanobody-Tn5 transposomes
40. Collect the eluate. Prepare the oligonucleotides and add the oligonucleotides to the eluate and incubate for 1h at 30 °C .

SECTION: Preparing GFP nanobody-Tn5 transposomes
41. Dialyze overnight in Tn5 dialysis buffer.
SECTION: Preparing GFP nanobody-Tn5 transposomes
42. Concentrate the protein using 30kDa Amicon centrifugation filter. Store the concentrated protein in Tn5 storage buffer, prepare aliquots, flash freeze in liquid nitrogen and store at -80 °C . 

SECTION: Preparing GFP nanobody-Tn5 transposomes
40.1. Resuspend oligonucleotides at 200 uM in water. 
SECTION: Preparing GFP nanobody-Tn5 transposomes
40.2. Incubate for 5 min at 95 °C in a thermocycler and cool down over