TITLE: Single cell CUT and Tag on 10x genomics platform
AUTHORS: Marek Bartosovic, Goncalo Castelo-Branco

[DESCRIPTION]
scCU&Tag on 10x platform uses scCUT&Tag protocol from Steven Henikoff's lab (Kaya-Okur et al., 2019), and scATAC-seq chromium platform (10x Genomics) to perform single-cell barcoding. The method can be used to obtain high quality data for tens of thousands of single cells with high specificity. 

See also accompanying BiorXiv preprint https://www.biorxiv.org/content/10.1101/2020.09.02.279703v1 for more details. 

scCUT&Tag can be performed on cell lines or freshly isolated cells from primary tissue. 



[STEPS]
SECTION: Nuclei preparation and primary antibody incubation
5. Dissociate your tissue/ cell line into a single-cell (single-nuclei) suspension and count your cells using manual counting chamber. We generally use between 150,000-250,000 cells as input from fresh samples and up to 500,000 nuclei extracted from frozen tissue.

If FACS sorting from a dissociated tissue, sort directly into eppendorf tubes with 500 ul of the antibody buffer cooled to 4 degrees. 
SECTION: Nuclei preparation and primary antibody incubation
6. Centrifuge cells/nuclei for 5 minutes at 300x g. Resuspend in 200 ul of Antibody buffer and centrifuge again 3 minutes at 600x g. 
 

SECTION: Nuclei preparation and primary antibody incubation
7. Prepare 1:50 dilution of primary antibody in 200 ul of antibody buffer per sample. 

Resuspend the pelleted nuclei in 100 ul of the antibody buffer
 

SECTION: Nuclei preparation and primary antibody incubation
8. Incubate the cells with primary antibody overnight at 4 degrees on rotating wheel or roller with slow rotation speed. 
  

SECTION: Secondary antibody incubation
9. Centrifuge the nuclei, 3minutes at 600x g.

Remove supernatant. Wash once with 200ul of Dig-wash buffer. 


SECTION: Secondary antibody incubation
10. Centrifuge 3 minutes at 600x g

Remove the supernatant. 
SECTION: Secondary antibody incubation
11. Prepare 200ul of 1:50  diluted secondary antibody per sample in Dig-Wash-BSA buffer. 

Resuspend the nuclei in 200 ul of diluted secondary antibody.    
 

SECTION: Secondary antibody incubation
12. Incubate 1 hour 60 min  rotating at room temperature.

SECTION: pA-Tn5 incubation
13. Centrifuge 3 minutes at 600x g

Remove the supernatant. Resuspend in 200 ul of Dig-300-BSA wash. 
SECTION: pA-Tn5 incubation
14. Repeat the wash-centrifugation 2 more times for total of 3 washes
SECTION: pA-Tn5 incubation
15. Prepare 200 ul of 1:100 diluted pA-Tn5 in Dig-300-BSA buffer per sample. 


SECTION: pA-Tn5 incubation
16. Resuspend the pellet in 200 ul of diluted pA-Tn5
SECTION: pA-Tn5 incubation
17. Incubate for 1 hour 60 min  rotating at room temperature

SECTION: Tagmentation
18. Centrifuge 3 minutes at 300x g

Remove the supernatant. Resuspend in 200 ul of Dig-300-BSA wash. 
SECTION: Tagmentation
19. Repeat the wash-centrifugation 2 more times for total of 3 washes
SECTION: Tagmentation
20. Resuspend the nuclei in 200ul of tagmentation buffer. Incubate 1hour 60 min at 37 degres in water bath or thermomixer. 

Tap the tubes 3-4 times during the incubation to prevent nuclei sedimentation
 

SECTION: Tagmentation
21. 
Prepare 1x Diluted nuclei buffer (DNB) supplemented with 2%BSA

Prepare STOP buffer by mixing 200ul of Dig-300 buffer with 10 ul of 500mM EDTA per sample.

 

 



SECTION: Tagmentation
23. Centrifuge for 3 minutes at 300x g.

Wash the nuclei with 200 ul of 1xDNB+BSA
SECTION: Counting the nuclei
31. Centrifuge the nuclei for 3 minutes at 300x g.

Resuspend the nuclei in 15-25ul of1xDNB+2%BSA
SECTION: Counting the nuclei
32. Count the nuclei using manual counting chamber. 

Use 2ul of Nuclei suspension + 8ul of trypan blue in 2 replicates
SECTION: Counting the nuclei
33. Calculate nuclei concentration and refer to the 10x scATAC-seq manual regarding concentration and volume required for chromium chip loading for desired yield. 

 

SECTION: qPCR cycle check (optional)
24. While nuclei are in 200ul of DNB, take 10% (20 ul) in a new tube, add 73 ul of water, 5 ul of 10%SDS and 2ul of proteinase K. Mix well by pipetting up and down 5x. 


Keep the remaining nuclei on ice for about 2 hours during optional cycle check. This does not influence efficiency of scCUT&Tag. 
SECTION: qPCR cycle check (optional)
25. Incubate for 30 minutes 30 min  at 50 degrees. 

SECTION: qPCR cycle check (optional)
26. Purify the DNA using ZYMO DNA Clean an Concentrator-5 kit. Use 1:5 ratio of binding buffer.
SECTION: qPCR cycle check (optional)
27. Elute the DNA in 25 ul of elution buffer.
SECTION: qPCR cycle check (optional)
28. Mix qPCR reaction:

Fw primer ATAC*           2ul
Rev primer  ATAC*         2ul
water                                8.5 ul
10xSYBR                          2.5 ul
2x NEBnext MM             25 ul
eluted DNA template    10ul

total                             50 ul

*Indexed primers described by Buenrostro, J.D. et al. Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 523:486 (2015).
SECTION: 10x scATAC-seq protocol
34. Skip Step 1 in the scATAC-seq manual, start at Step 2 GEM Generation and barcoding   

If using scATAC-seq kit v1.1, thaw ATAC buffer B

SECTION: 10x scATAC-seq protocol
35. Prepare master mix:


scATAC v1:


Nuclei suspension            15 ul
Barcoding reagent            61.5 ul
Reducing agent B            1.5 ul
Barcoding Enzyme            2 ul


scATAC v1.1:

Nuclei suspension            8 ul
ATAC buffer B                    7 ul
Barcoding reagent B         56.5 ul
Reducing agent B            1.5 ul
Barcoding Enzyme            2 ul
 

SECTION: 10x scATAC-seq protocol
36. Load the 10x chromium chip according to manufacturers instructions in the 10x scATAC-seq kit in Step 2
SECTION: qPCR cycle check (optional)
29. Run qPCR program: 

1. 72C 5min
2. 98C 1min
3. 98C 15s
4. 63C 10s (data collection)
5. GOTO 3 39x

SECTION: 10x scATAC-seq protocol
37. Continue with the protocol exactly as in manufacturers instructions in the 10x scATAC-seq from Step 3 onwards
SECTION: Buffers preparation
4.   

SECTION: Tagmentation
22. Add 200ul of STOP buffer and mix well by pipetting up and down several times. 

 

SECTION: Tn5 loading
1. Dilute Tn5ME-A, Tn5ME-B and Tn5ME-rev oligos to 100uM

Mix in two separate PCR tubes 
1. 10ul Tn5ME-A + 10ul Tn5ME-rev
2. 10ul Tn5ME-B + 10ul Tn5ME-rev

Denature on a thermocycler for 5 min at 95°C, and cool down slowly on the thermocycler by ramping down by 0.1C/s
SECTION: Tn5 loading
3. Mix Tn5 with the annealed oligonucleotides:

Tn5ME-A/Tn5ME-rev         2 ul
Tn5ME-B/Tn5ME-rev         2 ul
Glycerol                               21.56 ul
2x Dialysis buffer               21.3 ul
Tn5 (3.5 mg/ml)                 3.14 ul
total                                      50 ul

Mix gently with pipette and incubate for 1h at room temperature.
Store enzyme at -80C long term. Keep working stock at -20C. 

Test small batch of Tn5 and if the loading is successful, scale up the loading reaction. 
SECTION: Tn5 loading
2. Prepare 2x dialysis buffer:

100mM      HEPES-KOH pH7.2
200 mM     NaCl
0.2 mM      EDTA
2mM          DTT*
0.2%           Triton-X      
20%            Glycerol

* DTT is toxic and should only be added to the stock solution immediately prior to use (e.g. 45 ul buffer + 5 ul DTT)
Keep buffer at 4 °C
SECTION: qPCR cycle check (optional)
30. Typical successful experiment has CT value in < 15 and can be easily distinguished from failed runs where CT > 20. 
SECTION: 10x scATAC-seq protocol
38. For final PCR amplification, use standard chromium scATAC-seq kit amplification protocol, with 16-20 PCR cycles depending on the antibody.