TITLE: Nano-CUT&amp;Tag for multimodal profiling of the chromatin
AUTHORS: Marek  Bartosovic, Goncalo Castelo-Branco

[DESCRIPTION]
Nano-CUT&Tag is a multimodal technology to profile several histone modifications at the same with single-cell resolution. Nano-CUT&Tag implements a novel Tn5 fusion proteins to anti-mouse and anti-rabbit secondary nanobodies. Optionally, ATAC-seq can be performed prior on the same sample to profile open chromatin at the same time. Novel tagmentation protocol, which involves two-step tagmentation by MeA and MeB oligonucleotides yields increased number of fragments per cell comparing to previous single-cell CUT&Tag protocols on 10x Genomics platform. 

  
  




  

  





[BEFORE_START]
Before starting, make yourself familiar with the 10x genomics Chromium Single Cell ATAC Reagent Kits User Guide (v1.1 Chemistry). 

Follow all the best practices and tips given in the 10x genomics Chromium Single Cell ATAC Reagent Kits User Guide. 

This protocol is compatible with Chromium Next GEM Single Cell ATAC Library & Gel Bead Kit v1.1 has not been tested with Chromium Next GEM Single Cell ATAC Kit v2. 

  


[GUIDELINES]
This protocol involves large number of centrifugation steps, and it is critical to prevent nuclei loss and/or clumping. Therefore, always use swinging bucket rotor centrifuge for all centrifugation steps, include 2% BSA in all buffers which come in contact with nuclei and carefully aspirate the supernatant. It is better to leave cca 5-10 ul of supernatant in the tube rather than lose the nuclei. 

Clumping of nuclei can be a problematic, but generally, having cleaner nuclei helps to prevent clumping. Removing of debris from your sample is highly recommended. For mouse brain Debris Removal Solution has proven to be effective in removing excess debris (Miltenyi, 130-109-398). 

Some steps in this protocol involve usage of in-house produced Tn5/nano-Tn5. Individual tagmentation steps should be optimised according to specific batch of the proteins. 

The two-step tagmentation protocol can also be performed with pA-Tn5 protein. Load pA-Tn5 only with MeA/Me-Rev oligos and follow the protocol as described here. pA-Tn5 profiling should be compatible with ATAC-seq in the same sample, but this was not tested. 

[STEPS]
SECTION: Tn5 loading
1. Annealing adaptor sequences:

Tn5_MeA_P5_noBCD.         
5' TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG

Tn5_MeA_P5_bcdA.   	       
5'-TCGTCGGCAGCGTCTCCACGC TATAGCCT GCGATCGAGGACGGCAGATGTGTATAAGAGACAG

Tn5_MeA_P5_bcdB	       
5'-TCGTCGGCAGCGTCTCCACGC ATAGAGGC GCGATCGAGGACGGCAGATGTGTATAAGAGACAG

Tn5_MeA_P5_bcdC	       
5'-TCGTCGGCAGCGTCTCCACGC CCTATCCT GCGATCGAGGACGGCAGATGTGTATAAGAGACAG

Tn5ME-B:                               
5′- GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′

Tn5MErev.                             
5'-[phos]CTGTCTCTTATACACATCT-3'




 

 

 


SECTION: Tn5 loading
2. Resuspend the oligonucleotides (Tn5ME-A, Tn5ME-B, Tn5MErev) in water to a final concentration of 100µM each. 
SECTION: Tn5 loading
3. Mix equimolar amounts of Tn5MErev/Tn5ME-A and Tn5MErev/Tn5ME-B in separate 200 µl PCR tubes. 

e.g: 

tube1: 10 µL  Tn5ME-A + 10 µL  Tn5MErev
tube2: 10 µL  Tn5ME-B + 10 µL  Tn5MErev

SECTION: Tn5 loading
4. Denature in the thermocycler for 5 min  at 95 °C , and cool down slowly on the thermocycler by ramping down to 20 °C  by 0.1 °C 

Pause point: Store the annealed oligos at -20 °C 

SECTION: Tn5 loading
5. Mix annealing mix. 

The final Tn5 concentration is 2 micromolar (µM)  of Tn5 dimer

Use unique barcodes for specific nano-Tn5 or WT-Tn5. 

Mouse Nano-Tn5 (MeA/P5 loaded):

4 µL            Annealed, barcoded MeA/Me-Rev oligos 50 micromolar (µM)  (e.g. barcode A, Me-A/Me-Rev)
21 µL         Glycerol
3 µL           Nano-Tn5 (mouse, 5mg/ml, MW = 73941 g/mol; 67.6 micromolar (µM)  )
22 µL        2x Tn5 loading buffer


Rabbit Nano-Tn5 (MeA/P5 loaded):
 
4 µL              Annealed, barcoded MeA/Me-Rev oligos 50 micromolar (µM) (e.g. barcode B, Me-A/Me-Rev)
21 µL            Glycerol
2.2 µL           Nano-Tn5 (rabbit, 6.8mg/ml, MW = 73013 g/mol 93.1 micromolar (µM) )
22.8 µL        2x 2x Tn5 loading buffer


WT Tn5-MeA with barcode (for ATAC-seq)

4 µL              Annealed, barcoded MeA/Me-Rev oligos 50 micromolar (µM) (e.g. barcode C, Me-A/Me-Rev)
21 µL            Glycerol
3.1 µL           Tn5 (3.5 mg/ml, MW = 53300 g/mol 65.7 micromolar (µM) )
21.9 µL        2x 2x Tn5 loading buffer


WT Tn5-MeB unbarcoded (for 2nd tagmentation) 

 
4 µL              Annealed MeB/Me-Rev oligos 50 micromolar (µM) (un-barcoded, Me-B/Me-Rev)
21 µL            Glycerol 
3.1 µL           Tn5 (3.5 mg/ml, MW = 53300 g/mol 65.7 micromolar (µM) )
21.9 µL        2x Tn5 loading buffer



 

 


2x Dialysis buffer
100 mM HEPES-KOH pH7.2, 200 mM NaCl, 0.2 mM EDTA, 0.2% Triton-X, 20% Glycerol, Store at 4 °C ; 
Add DTT fresh to the 2x dialysis buffer just before loading (2mM final). Keep 200 mM DTT stock at -20 °C 

For details on buffer preparation see Materials section
SECTION: Tn5 loading
6. Incubate for 60 min  at Room temperature 

SECTION: Tn5 loading
7. Store the loaded nano-Tn5 at -20 °C 

SECTION: ATAC-seq (optional)
8. Dissociate tissues/cells of interest by desired method  and obtain single-cell suspension. Wash the cells once with 1x PBS


 


SECTION: ATAC-seq (optional)
9. Centrifuge the cells for 10 min  at 300x g at 4 °C . Discard the supernatant. 

SECTION: ATAC-seq (optional)
10. Add 200 µL of ATAC lysis buffer (for 200,000 cells, see materials section for buffer recipe). Pipette up and down gently 3x and incubate on ice for 3 min 


 

SECTION: ATAC-seq (optional)
11. Add 1 mL  of ATAC wash buffer and gently invert the tube 3x. 

SECTION: ATAC-seq (optional)
12. Centrifuge at 500x g for 10 min . Discard the supernatant. 

SECTION: ATAC-seq (optional)
13. Prepare transposition mix:


100 µL        2X TD Buffer (see materials section)
66 µL          1X PBS
2 µL            10% Tween-20 (final 0.1% v/v)
2 µL            1% Digitonin (final 0.01% v/v)
10 µL         Tn5 Transposase (loaded with uniquely barcoded oligonucleotides-MeA only) 
20 µL         nuclease-free H2O

SECTION: ATAC-seq (optional)
14. Resuspend the nuclei in 200 µL  of transposition mix (for 200,000 cells). 

 





SECTION: ATAC-seq (optional)
15. Incubate for 30 min  at 37 °C in thermomixer at 1000 rpm. 

SECTION: ATAC-seq (optional)
16. Stop the tagmentation by adding 10 µL of 500 mM EDTA. Mix by pipetting up and down 3x. 

SECTION: ATAC-seq (optional)
17. Centrifuge for 10 min at 500x g

SECTION: ATAC-seq (optional)
18. Remove the supernatant. Resuspend in 200 µL  of CUT&Tag Antibody buffer.

SECTION: ATAC-seq (optional)
19. Centrifuge for 3 min at 600x g. Remove the supernatant. 

Proceed to CUT&Tag Antibody binding (Step 23). 

SECTION: CUT&amp;Tag Nuclei isolation (nano-CUT&amp;Tag without ATAC)
20. Dissociate tissues/cells of interest by desired method and extract nuclei by incubation for 3 min  in 200 µL of Antibody buffer on ice. 


 

 

SECTION: CUT&amp;Tag Nuclei isolation (nano-CUT&amp;Tag without ATAC)
21. Centrifuge the nuclei for 3 min at 600x g. 

SECTION: CUT&amp;Tag Nuclei isolation (nano-CUT&amp;Tag without ATAC)
22. Remove the supernatant. 
 



      

SECTION: Antibody binding
23. Prepare antibody mix

Starting concentrations (can be further optimised, depending on the antibody)


1:100 primary antibody 
1:100 nano-Tn5

Final volume 100ul per sample. 
SECTION: Antibody binding
24. Resuspend the nuclei pelet in the prepared antibody mix (1:100 primary antibody, 1:100 nano-Tn5) by pipetting up and down 5x. 
SECTION: Antibody binding
25. Incubate 3 min on with rotation on a horizontal roller at 4 °C 
  

 

SECTION: Washing and Tagmentation
26. The next day, centrifuge for 3 min  at 600xg. 

SECTION: Washing and Tagmentation
27. Remove the supernatant and resuspend in 200 ul of Dig-300 wash buffer. 


 

SECTION: Washing and Tagmentation
28. Repeat the steps 27-28 for total of 2 washes. 
SECTION: Washing and Tagmentation
29. Resuspend the nuclei pellet in 200 ul of Tagmentation buffer. Pipette mix 5x to resuspend the pellet. 
SECTION: Washing and Tagmentation
30. Incubate for 60 min  at 37 °C 


 

SECTION: Washing and Tagmentation
31. During the incubation prepare tagmentation STOP buffer.

Prepare 2x diluted nuclei buffer (DNB) from 20x nuclei buffer (10x scATAC-seq, PN: 2000207). Store the aliquots of 2x DNB at -20 °C 

Stop buffer:

100 µL    2x Diluted Nuclei buffer 
20 µL      20% BSA
70 µL      water
10 µL      500 mM EDTA). 


1x DNB + BSA buffer:

500 µL    2x Diluted Nuclei buffer (10x scATAC-seq kit, dilute down from 20x (PN: 2000207)) 
100 µL      20% BSA
400 µL      water


SECTION: Washing and Tagmentation
32. Stop the tagmentation by removing from 37 thermoblock and adding  200 µL  of STOP buffer. Mix well by pipetting up and down 3x. 

SECTION: Washing and Tagmentation
33. Centrifuge for 3 min  at 300x g. Remove supernatant

SECTION: Washing and Tagmentation
34. Resuspend the nuclei in 200 µL  of 1x DNB+BSA buffer. 

SECTION: Washing and Tagmentation
35. Centrifuge for 3 min  at 300x g. Remove supernatant

SECTION: Washing and Tagmentation
36. Resuspend the nuclei in 200 µL  of 1x DNB+BSA buffer. 

SECTION: Washing and Tagmentation
37. Centrifuge for 3 min  at 300x g. 

SECTION: Washing and Tagmentation
38. Remove the most of the supernatant, leave the nuclei in cca 10 µL of remaining 1xDNB+BSA buffer.

SECTION: Washing and Tagmentation
39. Add 10 µL  of 1xDNB+BSA buffer, for final 20 ul of nuclei suspension. Measure the exact volume using P20 pipette. 

SECTION: Nuclei counting )
40. Count the concentration of nuclei. Use 2 µL of nuclei suspension and mix with 8 µL  of trypan blue. For counting use manual counting chambers (VWR, 630-1893). Do the counting in two replicates for more accuracy. 

  

SECTION: Nuclei counting )
41. To aim for cca 5,000 recovered nuclei, load cca 16,000 nuclei or determine ratio of loaded/recovered nuclei empirically. 


 

SECTION: Chromium Next GEM barcoding
42. 
 

 
  

SECTION: Chromium Next GEM barcoding
43. Mix the GEM generation and barcoding mix. Use the desired volume of nuclei and fill up to 15 µL  with 1x DNB+BSA buffer. 


Prepare nuclei mix and keep it on ice:


7 µL                 ATAC buffer B
X µL                 Nuclei (Use =< 8ul of nuclei)
up to 15 µL    1x DNB+BSA buffer

keep on ice
SECTION: Chromium Next GEM barcoding
44. Prepare barcoding mix and : 

56.5 µL                         Barcoding reagent B
1.5 µL                            Reducing agent B
2 µL                                Barcoding enzyme

keep it on ice
SECTION: Chromium Next GEM barcoding
45. Assemble the Chromium Next GEM Chip H according to the manufacturer's instructions. 

  




SECTION: Chromium Next GEM barcoding
46. Load the Chromium Next GEM Chip H according to the manufacturer's instructions. Run the droplet generation.  

  
  

SECTION: Chromium Next GEM barcoding
47. Recover droplets from the Next GEM Chip H according to the manufacturer's instructions. 

  

SECTION: Chromium Next GEM barcoding
48. Incubate in PCR cycler according to the manufacturer's instructions. 

The linear amplification (LA) and single-cell barcoding occurs at this step. 

  

SECTION: Post-GEM incubation cleanup
49. Perform post-GEM incubation cleanup according to manufacturer's instructions - Steps 3.1-3.2
  

  

  

  

SECTION: Post-GEM incubation cleanup
50. Use 2ul of the purified DNA to measure the concentration (optional) using Qubit high sensitivity dsDNA kit. 

 

 

SECTION: MeB tagmentation and library preparation
51. Mix MeB tagmentation reaction:

38-40 µL            Barcoded LA product (from previous step, typically 5-10 ng)
50 µL             2x TD buffer 
0.5 µL           MeB-loaded Tn5 (starting point, should be adjusted for specific batch of Tn5 and yield of DNA)
up to 100 µL        Water

Incubate in PCR cycler 30 min  at 37 °C  ->  4 °C  hold
Heated lid at 50 °C

SECTION: MeB tagmentation and library preparation
52. Purify the DNA using Zymo DNA Clean and concentrator-5 kit according to manufacturer's instructions. 

Transfer the sample into 1.5 ml eppendorf tube. 
Add 500 µL  of zymo binding buffer to your sample. 
Wash 2x with 200 µL of zymo wash buffer. 
Perform one more dry spin 1min at max speed to remove residual liquid.

Elute the DNA in 40 µL  of DNA elution buffer (Zymo kit) . Incubate 2minutes on column, then centrifuge. 

  



SECTION: MeB tagmentation and library preparation
53. Run PCR library amplification:



40 µL           Purified DNA from previous step (sample)
7.5 µL          SI-PCR primer B (10x ATAC-seq kit; PN: 2000128)
2.5 µL          Individual Single Index N Set A primer (product code: 1000212)
50 µL           AMP mix (10x ATAC-seq kit; PN: 2000047/ 2000103)

Incubate in PCR cycler with the following program:

Lid temperature 105 °C , volume 100 µL .

1. 72 °C     5min
2. 98 °C     45sec
3. 98 °C    20sec    |
4. 67 °C    30sec    | Repeat 13x
5. 72 °C    20sec    |
6. 72 °C    1min
7. 4 °C       hold

 


SECTION: MeB tagmentation and library preparation
54. Purify the final library using according to Step 4.2 in Chromium Next GEM Single Cell ATAC Reagent Kits v1.1.   

SECTION: Sequencing preparation
55. Sequence on Illumina NovaSeq v1.5 platform with read setup : 36-8-48-36 (R1-I1-I2-R2) using custom sequencing primers: 

Custom_primer_R1: GCGATCGAGGACGGCAGATGTGTATAAGAGACAG
Custom_primer_I2:    CTGTCTCTTATACACATCTGCCGTCCTCGATCGC


R2 & I1 standard
SECTION: Sequencing preparation
56. Library structure:

P5 side:

  

P7 side: 

  

SECTION: Sequencing preparation
57. We typically aim for ~25,000 read pairs per cell, meaning 125,000,000 reads for 5000 cells (~one 10x lane). 
SECTION: Desired outcome
58. Typical bioanalyzer trace of a successful experiment shows good and even  distribution of fragment sizes and is not overtagmented or undertagmented. Both over- and under- tagmenation will lead to reduced complexity of the library.