TITLE: CUT&amp;Tag-direct for whole cells with CUTAC 
AUTHORS: Steven Henikoff, Jorja Henikoff, Kami Ahmad

[DESCRIPTION]
  
We previously introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling method in which antibody tethering of the Tn5 transposase to a chromatin epitope of interest maps specific chromatin features in small samples and single cells.With CUT&Tag, intact cells or nuclei are permeabilized, followed by successive addition of a primary antibody, a secondary antibody, and a chimeric Protein A-Transposase fusion protein that binds to the antibody. Addition of Mg++ activates the transposase and inserts sequencing adapters into adjacent DNAin situ.We have since adapted CUT&Tag to also map chromatin accessibility by simply modifying thetransposaseactivation conditions when using histone H3K4me2, H3K4me3 or Serine-5-phosphorylated RNA Polymerase II antibodies. Using these antibodies, tagmentation of accessible DNA sites is redirected to produce chromatin accessibility maps with exceptionally high signal-to-noise and resolution. All steps from nuclei to amplified sequencing-ready libraries are performed in single PCR tubeswith non-toxic reagents using inexpensive equipment, making our simplified strategy for simultaneous chromatin profiling and accessibility mapping suitable for the lab, home workbench or classroom. 

New with this version are simple modifications to make the one-tube CUT&Tag-direct protocol suitable for whole cells, while providing data quality comparable to that for Bench-top CUT&Tag.




[BEFORE_START]
Optional: Prepare and cryopreserve whole cells (Version 4 only) or nuclei (STEPS 52-64 at the lab)
Prepare reagents (STEP 1, OK at the lab, at home or in the classroom)

[GUIDELINES]
Based on https://www.protocols.io/view/bench-top-cut-amp-tag-z6hf9b6.

[STEPS]
SECTION: REAGENT SETUP (for up to 16 samples)
1. Binding buffer Mix 200 μL 1M HEPES-KOH pH 7.9*, 100 μL 1M KCl, 10 μL 1M CaCl2 and 10 μL 1M MnCl2, and bring the final volume to 10 mL with dH2O. Store the buffer at 4 °C for up to several months. *HEPES-NaOH pH 7.5 is OK.

Triton-Wash buffer Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 12.5 μL 2 M spermidine and 250 µl 10% Triton-X100, bring the final volume to 50 mL with dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store the buffer at 4 °C for up to 2 days.

Antibody buffer Mix 5 µl 200X BSA and 4 µl 0.5 M EDTA with 1 ml Triton-Wash buffer and chill on ice. BSA is present in some but not all antibody solutions, and 0.1% BSA in this buffer helps to prevent bead loss during later steps.

Triton-300-wash buffer Add 750 µL 5M NaCl to 24.3 ml Triton-Wash buffer. Store at 4 °C for up to 2 days.

CUT&Tag Tagmentation buffer Mix 1 mL Triton-300-wash buffer and 10 µL 1 M MgCl2 (to 10 mM).

CUTAC-DMF Tagmentation buffer Mix 780 µl dH2O, 200 µl N,N-dimethylformamide, 10 µl 1 M TAPS pH 8.5, 5 µl Triton-X100 and 5 µl 1 M MgCl2 (10 mM TAPS, 5 mM MgCl2, 20% DMF, 0.05% Triton-X100). Store the buffer at 4 °C for up to 1 week.

TAPS wash buffer Mix 1 mL dH2O, 10 µL 1 M TAPS pH 8.5, 0.4 µL 0.5 M EDTA (10 mM TAPS, 0.2 mM EDTA).

1% SDS-ProtK Release solution (For 32 samples) Mix 20 µl 10% SDS and 2 µl 1 M TAPS pH 8.5 in 178 µl dH2O. Just before use add 20 µL Thermolabile Proteinase K (NEB cat. no. P8111S).

6% Triton neutralization solution Mix 600 µL 10% Triton-X100 + 400 µL dH2O.
SECTION: Prepare Concanavalin A-coated beads (15 min)
2. Resuspend and withdraw enough of the ConA bead slurry, ensuring that there will be 5 μl for each final sample of up to ~50,000 mammalian cells, which yield ≥50% K562 nuclei using this protocol. Transfer the ConA bead slurry into 1 mL of Binding buffer in a 1.5 mL tube.
 

SECTION: Prepare Concanavalin A-coated beads (15 min)
3. Mix by pipetting. Place the tube on a magnet stand to clear (~1 min).
SECTION: Prepare Concanavalin A-coated beads (15 min)
4. Withdraw the supernatant completely, and remove the tube from the magnet stand. Add 1 mL Binding buffer and mix by pipetting up and down.
SECTION: Prepare Concanavalin A-coated beads (15 min)
5. Place on the magnet stand to clear, remove and discard the supernatant, and resuspend in 80 µL Binding buffer (5 μL per sample).
SECTION: Binding whole cells to ConA beads
6. Thaw a frozen aliquot of cells at room temperature, for example by placing in a 20 ml beaker of water.

 

 

SECTION: Binding whole cells to ConA beads
7. Transfer the thawed cell or nuclei suspension in aliquots of up to 60,000 starting mammalian cells to each thin-wall PCR tube and mix with 5 µL ConA beads. Attach to tube Rotator and rotate at room temperature for 10 min.

 

 

SECTION: Binding whole cells to ConA beads
8. Place the tubes on the magnet stand to clear and withdraw the liquid.
 

SECTION: Bind primary antibody (1 hr)
9. For each CUT&Tag and CUTAC sample, mix the primary antibody 1:25-1:100 with Antibody buffer. Resuspend beads in 25 µL per sample with gentle vortexing.
 

 

SECTION: Bind primary antibody (1 hr)
10. Place on a rotator at room temperature and incubate 1-2 hr. 

60 min 

 

 

 

SECTION: Bind secondary antibody (1 hr)
11. Place tubes on the magnet stand to clear and discard the supernatant.
 

SECTION: Bind secondary antibody (1 hr)
12. Mix the secondary antibody 1:100 in Triton-Wash buffer and squirt in 25 µL per sample while gently vortexing to allow the solution to dislodge the beads from the sides. 
 

SECTION: Bind secondary antibody (1 hr)
13. Place the tubes on a rotator and rotate at room temperature for 0.5-1 hr.30 min
SECTION: Bind secondary antibody (1 hr)
14. After a quick spin (<500 x g or just enough to remove the liquid from the sides of the tube), place the tubes on the magnet stand to clear and remove and discard the supernatant with two successive draws, using a 20 µL tip with the pipettor set for maximum volume. 

SECTION: Bind secondary antibody (1 hr)
15. With the tubes still on the magnet stand, carefully add 500 µL of Triton-Wash buffer. The surface tension will cause the beads to slide up along the side of the tube closest to the magnet.
SECTION: Bind secondary antibody (1 hr)
16. Slowly remove 470 µL of supernatant with a 1 mL pipette tip without disturbing the beads.
 

SECTION: Bind secondary antibody (1 hr)
17. After a quick spin (< 500 x g or just enough to remove the liquid from the sides of the tube), place the tubes back into the magnet stand and remove the remaining supernatant with a 20 uL pipettor multiple times if necessary, to remove the entire supernatant without disturbing the beads. Proceed immediately to the next step.
SECTION: Bind pA-Tn5 adapter complex (1.5 hr)
18. Mix pAG-Tn5 pre-loaded adapter complex in Triton-300-wash buffer following the manufacturer's instructions (e.g. 1:20 for Epicypher CUTANA).


SECTION: Bind pA-Tn5 adapter complex (1.5 hr)
19. Pipette in 25 µL per sample of the pA-Tn5 mix while vortexing and invert by rotation to ensure that beads adhering to the sides near the top of the top are resuspended. 
 

SECTION: Bind pA-Tn5 adapter complex (1.5 hr)
20. After a quick spin, place the tubes on a rotator at room temperature for 1-2 hr.60 min
SECTION: Bind pA-Tn5 adapter complex (1.5 hr)
21. After incubating in the rotator, perform a quick spin and place the tubes in the magnet stand.
SECTION: Bind pA-Tn5 adapter complex (1.5 hr)
22. Carefully remove the supernatant using a 20 µL pipettor twice to avoid disturbing the beads as in Step 16.

SECTION: Bind pA-Tn5 adapter complex (1.5 hr)
23. 1.With the tubes still on the magnet stand, add 500 µl of the Triton-300-wash buffer.
SECTION: Bind pA-Tn5 adapter complex (1.5 hr)
24. Slowly withdraw 470 µl with a 1 ml pipette tip without disturbing the beads as in Step 16.
SECTION: Bind pA-Tn5 adapter complex (1.5 hr)
25. After a quick spin, place the tubes back on the magnet stand and remove and discard the supernatant with a 20 µL pipettor using multiple draws. Proceed immediately to Step 27. For a CUTAC sample proceed immediately to Step 26.
SECTION: CUTAC Tagmentation (performed in parallel with other samples)
26. Resuspend the bead/nuclei pellet in 50 µL CUTAC tagmentation solution (5 mM MgCl2, 10 mM TAPS) while vortexing or inverting by rotation to allow the solution to dislodge most or all the beads as in Step 19. Place the tube(s) in a cold block and proceed to Step 28.
 

SECTION: CUT&amp;Tag tagmentation and particle release (3.5 hr)
27. Resuspend the bead/nuclei pellet in 50 µL tagmentation solution while vortexing or inverting by rotation to allow the solution to dislodge most or all the beads as in Step 19.
SECTION: CUT&amp;Tag tagmentation and particle release (3.5 hr)
28. After a quick spin (<500 x g), incubate at 37 ºC for 1 hr (20 min for CUTAC) in a PCR cycler with a heated lid. Hold at 8 ºC. 60 min 20 min
SECTION: CUT&amp;Tag tagmentation and particle release (3.5 hr)
29. Place tubes on a magnet stand and remove and discard the supernatant with a 20 µL pipettor using multiple draws then resuspend the beads in 50 µL TAPS wash and invert by rotation as in Step 19.
SECTION: CUT&amp;Tag tagmentation and particle release (3.5 hr)
30. After a quick spin, place tubes on the magnet stand, and withdraw the liquid with a 20 µL pipettor using multiple draws.
SECTION: CUT&amp;Tag tagmentation and particle release (3.5 hr)
31. Resuspend the beads in 5 µL 1% SDS-ProtK Release solution using a fresh 20 µL pipette tip to dispense while wetting the sides of the tubes to recover the fraction of beads sticking to the sides.
 

SECTION: CUT&amp;Tag tagmentation and particle release (3.5 hr)
32. After a quick spin, incubate at 37 ºC for 1 hr then 58 ºC for 1 hr in a PCR cycler with a heated lid to release pA-Tn5 from the tagmented DNA followed by inactivation of the Proteinase K.  120 min
SECTION: PCR (1 hr)
33. To the PCR tube containing the bead slurry add 15 µL of 6% Triton neutralization solution + 2 µL of 10 µM Universal or barcoded i5 primer + 2 µL of 10 µM uniquely barcoded i7 primers, using a different barcode for each sample. Vortex on full speed and place tubes in the metal tube holder on ice.

 

SECTION: PCR (1 hr)
34. Add 25 µL NEBnext (non-hot-start), vortex to mix, and perform a quick spin. Place the tubes immediately in the thermocycler and proceed immediately with the PCR.
SECTION: PCR (1 hr)
35. Begin the cycling program with a heated lid on the thermocycler:
    Cycle 1: 58 °C for 5 min (gap filling)
    Cycle 2: 72 °C for 5 min (gap filling)
    Cycle 3: 98 °C for 5 min
    Cycle 4: 98 °C for 10 sec
    Cycle 5: 60 °C for 10 sec
    Repeat Cycles 4-5 11 times
    72°C for 1 min and hold at 8 °C


 

 

 

 

SECTION: Post-PCR Clean-up (30 min)
36. After the PCR program ends, remove tubes from the thermocycler and add 65 µL of SPRI beads (ratio of 1.3 µL of SPRI beads to 1 L of PCR product). Mix by pipetting up and down.
SECTION: Post-PCR Clean-up (30 min)
37. Let sit at room temperature 5-10 min.5 min
SECTION: Post-PCR Clean-up (30 min)
38. Place on the magnetic stand for a few minutes to allow the solution to clear.
SECTION: Post-PCR Clean-up (30 min)
39. Remove and discard the supernatant.
SECTION: Post-PCR Clean-up (30 min)
40. Keeping the tubes in the magnetic stand, add 200 uL of 80% ethanol.
SECTION: Post-PCR Clean-up (30 min)
41. Completely remove and discard the supernatant.
SECTION: Post-PCR Clean-up (30 min)
42. Repeat Steps 40 and 41.
SECTION: Post-PCR Clean-up (30 min)
43. Perform a quick spin and remove the remaining supernatant with a 20 uL pipette, avoiding air drying the beads by proceeding immediately to the next step.
SECTION: Post-PCR Clean-up (30 min)
44. Remove from the magnet stand, add 22 µL 10 mM Tris-HCl pH 8 and vortex at full speed. Let sit for 5 min to 1 hr. 5 min
SECTION: Post-PCR Clean-up (30 min)
45. Place on the magnet stand and allow to clear.
SECTION: Post-PCR Clean-up (30 min)
46. Remove the liquid to a fresh 1.5 ml tube with a pipette, avoiding transfer of beads.
SECTION: Tapestation analysis and DNA sequencing (outsource)
47.  Determine the size distribution and concentration of libraries by capillary electrophoresis using an Agilent 4200 TapeStation with D1000 reagents or equivalent.

    
 

SECTION: Tapestation analysis and DNA sequencing (outsource)
48. Mix barcoded libraries to achieve equal representation as desired aiming for a final concentration as recommended by the manufacturer. After mixing, perform an SPRI bead cleanup if needed to remove any residual PCR primers.   

SECTION: Tapestation analysis and DNA sequencing (outsource)
49. Perform paired-end Illumina sequencing on the barcoded libraries following the manufacturer’s instructions. For maximum economy, paired-end PE25 is more than sufficient for mapping to large genomes.
 

SECTION: Data processing and analysis
50. Align paired-end reads to hg19 using Bowtie2 version 2.3.4.3 with options: --end-to-end --very-sensitive --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. For mapping E. coli carry-over fragments, we also use the --no-overlap --no-dovetail options to avoid possible cross-mapping of the experimental genome to that of the carry-over E. coli DNA that is used for calibration. Tracks are made as bedgraph files of normalized counts, which are the fraction of total counts at each basepair scaled by the size of the hg19 genome.
 

 
 
 

SECTION: Data processing and analysis
51. Our CUT&Tag Data Processing and Analysis Tutorial on Protocols.io provides step-by-step guidance for mapping and analysis of CUT&Tag sequencing data. Most data analysis tools used for ChIP-seq data, such as bedtools, Picard and deepTools, can be used on CUT&Tag data. Analysis tools designed specifically for CUT&RUN/Tag data include the SEACR peak caller also available as a public web server and CUT&RUNTools.
 

 

SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
52. Prepare NE1 buffer: Mix 1 ml 1M HEPES-KOH pH 7.9, 500 μL 1M KCl, 12.5 μL 2 M spermidine, 500 µL 10% Triton X-100, and 10 ml glycerol in 38 ml dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free.

Prepare Wash buffer Mix 1 mL 1 M HEPES pH 7.5, 1.5 mL 5 M NaCl, 12.5 μL 2 M spermidine, bring the final volume to 50 mL with dH2O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet.

Other reageants:
Phosphate-buffered saline (1X PBS, 10X stock solution from Fisher cat. no. BP3994)
16% (w/v) formaldehyde (10 x 1 ml ampules, Thermo-Fisher ca. no. 28906)
1.25 M glycine
Dimethyl sulfoxide (DMSO; Sigma-Aldrich cat. no. D4540)
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
53. Harvest fresh culture(s) in a conical centrifuge tube (15 ml or 50 ml) at room temperature and count cells.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
54. Centrifuge for 3 min at 600 x g in a swinging bucket rotor at room temperature and drain liquid.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
55. Resuspend in 1 volume of PBS at room temperature by pipetting up and down.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
56. Centrifuge for 3 min at 600 x g in a swinging bucket rotor at room temperature and drain the liquid.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
57. Resuspend in 1/2 volume (relative to starting culture) ice-cold NE1 buffer with gentle vortexing. Let sit on ice for 10 min.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
58. Centrifuge for 4 min at 1300 x g at 4 °C in a swinging bucket rotor and drain liquid by pouring off and inverting onto a paper towel for a few seconds.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
59. Resuspend in 1/2 volume of PBS. For unfixed nuclei, skip to Step 62.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
60. While gently vortexing, add 16% formaldehyde to 0.1% (e.g., 62 µL to 10 ml) and incubate at room temperature for 2 min.
 

 

SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
61. Stop cross-linking by adding 1.25 M glycine to twice the molar concentration of formaldehyde (e.g., 600 µL to 10 ml).
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
62. Centrifuge for 4 min at 1300 x g at 4 °C and drain liquid by pouring off and inverting onto a paper towel for a few seconds.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
63. Resuspend in Wash buffer to a concentration of ~1 million cells per ml. Check nuclei using ViCell or a cell counter slide.
SECTION: Appendix: Prepare and (optionally) lightly fix nuclei and cryopreserve (1 hr in the lab)
64. Nuclei may be slowly frozen by aliquoting 900 µL into cryogenic vials containing 100 µL DMSO, mixed well, then placed in a Mr. Frosty container filled to the line with isopropanol and placed in a -80 °C freezer overnight and stored at -80 °C long term.