TITLE: ATAC-seq from nuclei from frozen tissue
AUTHORS: Eric RA Pederson

[DESCRIPTION]
Modified ATAC-seq method for frozen tissue, in this case brain tissue.

[BEFORE_START]
The pre-experimentation steps are important 
Also it is important that all the primers have been ordered and reconstituted beforehand.

[GUIDELINES]
The Tn5 enzyme used in this experiment was from a locally produced batch from the  Protein Science Facility at the Karolinska Institutet in Stockholm, which is first discussed in Picelli and others (2014). Thus, if the Tn5 is purchased through a company it may react differently.

[STEPS]
SECTION: Pre-experimentation
1. All steps should be performed on ice or at 4 °C. 
SECTION: Nuclei extraction and filtration
6. Materials for the cutting of tissue on dry ice.
SECTION: Nuclei extraction and filtration
9. Place 20 mg frozen tissue into a pre-chilled 2 ml Dounce containing 1 ml cold 1x HB and let thaw for 5 min. 
SECTION: Pre-experimentation
2. Pre-chill all Dounces and pestles to 4 °C in a fridge or on ice

SECTION: Pre-experimentation
3. Pre-chill all tubes.

For each sample you are processing, you will need: 
(i) One 2 mL    per sample
(ii) Three   per sample
(iii) one PCR tube per sample 
(iv) One   for filtration step per sample
SECTION: Pre-experimentation
4. Prepare buffers. 
i) 6x Homogenization Buffer Stable Solution.
ii) 6x Homogenization Buffer Unstable Solution.
iii) 1x Homogenization Buffer Unstable Solution.
iv) ATAC-RSB
v) ATAC-RSB + 10% Tween 
vi) 2X TD buffer
vii) 2X DF buffer

SECTION: Pre-experimentation
4.1. i) 6x Homogenization Buffer stable Solution.
 
      		Amount 		Reagents 	  		2.27084 ml 		6x Homogenization Buffer stable 	  		3.78 ml 		100mM PMSF 	  		0.28 ul 		14.3 M B-mercaptoethanol 	  		1/2 tablet 		Protease inhibitor (cOmplete Mini) 	 

SECTION: Pre-experimentation
4.2. ii) 6x Homogenization Buffer Unstable Solution.
 
      		Amount 		Reagents 	  		2.27084 ml 		6x Homogenization Buffer stable 	  		3.78 ml 		100mM PMSF 	  		0.28 ul 		14.3 M B-mercaptoethanol 	  		1/2 tablet 		Protease inhibitor (cOmplete Mini) 	 
 
  

SECTION: Pre-experimentation
4.3. ii) 1x Homogenization Buffer Unstable Solution.
 
      		Amount 		Reagents 	  		2.166645 ml  		6x Homogenization Buffer Unstable Solution  	  		4.16 ml  		1 M sucrose  	  		2.6 ul  		500 mM EDTA  	  		130 ul  		10.00% NP10 	  		6.540755 ml 		H2O 	 

SECTION: Pre-experimentation
5. Tn5 assembly reaction
SECTION: Pre-experimentation
5.1. 
 


 

95 °C to 25 °C 
cooling -0.1°C/second

~1 hour on PCR machine
Olgio Annealing program


SECTION: Pre-experimentation
5.2. TN5 Assembly


 

Room temperature 60 min 


Tn5 from the Protein Science Facility at the Karolinska Institutet in Stockholm (Picelli et al.,2014)
or
 

SECTION: ATAC-seq
21. Add the calculated amount of cells into a2 mL lobind eppendorf tube with 1 ml ATAC-RSB + 10% Tween.

So taking the example above one would take 1.10 ul of nuclei in the 200 µL ATAC-RSB + 10% Tween to the new tube.

SECTION: ATAC-seq
22. Centrifuge nuclei for 10 min 900 rcf, 10 min, 4 °C 

SECTION: ATAC-seq
23. Discard most of the supernatant. Leave a bit in the bottom to ensure that there is enough for the reaction and that the pellet stays intact
SECTION: ATAC-seq
24. Add the reaction mix to the pellet and remaining water and resuspend the pellet in the mix


 

SECTION: ATAC-seq
25. Put the tube into the thermomixer at  
SECTION: ATAC-seq
26. Take the tubes out and immediately proceed to the column clean up
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27. Use the Zymo DNA clean & concentrator to clean the nuclei
 

SECTION: ATAC-seq
28. Can stop here and start the next day if necessary.
Leave samples in 4°
SECTION: Nuclei extraction and filtration
10. Dounce with “A” loose pestle until resistance goes away (~10 strokes).

Put the A pestle into the beaker of water
SECTION: Nuclei extraction and filtration
11. Dounce with “B” tight pestle for 20 strokes.

Put the B pestle into the beaker of water
SECTION: Nuclei extraction and filtration
12. Pour everything from the dounce into a 30 um MACS smartstrainer which is sitting on top of a labelled 50 ml falcon tube sitting in ice.
 

SECTION: Nuclei extraction and filtration
13. Let it drip through for 10-15 minutes.
SECTION: Nuclei extraction and filtration
7. In the most sterile way possible, cut a small piece of tissue, half a pea size or so, and leave it in the petri dish with a marking on the lid, in the dry ice. Weigh it and cut again if needed.

Make sure to use the ethanol to clean everything and be careful not to cut yourself.
SECTION: Nuclei extraction and filtration
6.1. - Gloves
- White warm gloves
- Dry ice
- Blades and handle
- Forceps
- Ethanol spray
- Cell culture dish 
SECTION: Nuclei extraction and filtration
8. Add 2 mL 1X HB buffer into the dounce, which is sitting in the ice.

Add  0.2 µL 1 M DTT and and 1 µL 100X Halt protease inhibitor 

SECTION: Nuclei extraction and filtration
14. Transfer to a labelled 2 mL Lobind Eppendorf tube, already cold from sitting in ice.

SECTION: Nuclei extraction and filtration
15. To pellet the nuclei, centrifuge 900 rpm, 10 min, 4 °C 

SECTION: Nuclei extraction and filtration
16. Discard supernatant
SECTION: Nuclei extraction and filtration
17. Resuspend the nuclei in 200 µL of ATAC-RSB + 10% Tween

SECTION: Nuclei extraction and filtration
18. Count the amount of cells using the cell counter in the cell lab.


SECTION: Nuclei extraction and filtration
19. Calculate the amount of nuclei to use for the next step;
(500000 nuclei)
SECTION: Nuclei extraction and filtration
20. Turn on the thermomixer to 37 °C 

SECTION: Nuclei extraction and filtration
18.1. Put 10 µL Trypton blue onto a piece of parafilm and mix with 10 µL of the sample

SECTION: Nuclei extraction and filtration
18.2. Take 10 µL of the mix and pipette into a cell counting cell

SECTION: Nuclei extraction and filtration
18.3. Measure on the cell counting machine, making sure to adjust for the smaller size of the nuclei.

The machine will think they are dead cells.
SECTION: Nuclei extraction and filtration
19.1. ATAC-seq requires 50,000 cells for the experiment. So for example if the number of nuclei is;

4.76 x 107

then the calculation will look like this;

50000 / 47600 = 1.10

SECTION: ATAC-seq
22.1. Here one can also save the nuclei for later. Simply spin the tube down with the new tube of diluted nuclei, discard the supernatant, add 300 µL  of the and put in the -20° freezer.

 10 min 900 rcf, 10 min, 4 °C 
SECTION: ATAC-seq
27.1. Add 2-7 volumes of the DNA binding buffer to each volume of DNA sample

So in this case the volume is approximately 50 µL , so add 300 µL of DNA binding buffer

SECTION: ATAC-seq
27.2. Transfer mixture to a provided Zymo-spin column in a collection tube
SECTION: ATAC-seq
27.3. Centrifuge 10000 x g, 30 s 
Discard supernatant
SECTION: ATAC-seq
27.4. Add 200 µL DNA wash buffer to the column

Centrifuge 10000 x g, 30 s 

SECTION: ATAC-seq
27.5. repeat the wash step
SECTION: ATAC-seq
27.6. Trasnfer the column to a 1.5 low-bind eppendorf tube.

Add 21 µL 2 DNA Elution buffer to the column.

Centrifuge 10000 x g, 30 s 

SECTION: ATAC-seq
29. Set up PCR;


 


 

SECTION: ATAC-seq
31. Proceed immediately to the qPCR amplification to determine additional cycles step
SECTION: ATAC-seq
30. Remove PCR tubes from the machine and put immediately onto ice.

SECTION: ATAC-seq
29.1. PCR program - ATAC-seq pre-amplification

 

SECTION: ATAC-seq
32. qPCR amplification to determine additional cycles

prepare a mix or master mix


 


 

 

SECTION: ATAC-seq
33. Determine the required amount of cycles that each sample needs in addition
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32.1. qPCR program


 

SECTION: ATAC-seq
32.2. qPCR extra setup options

- 6 machine not 6
- standard
- SYBR green

- no melt curve

- turn off ROX 

- Fill in sample list


SECTION: ATAC-seq
34. Continue the PCR with the additional amount of cycles skipping the beginning parts of the program
SECTION: ATAC-seq
35. Take the tubes out and immediately proceed to the column clean up
SECTION: ATAC-seq
33.1. Looking at the final amplification curve, determine the max fluorescence where the graph plateaus
SECTION: ATAC-seq
33.2. Determine 1/3 of that number.

For example if the max is 1400000 then 1/3 would be 433333


SECTION: ATAC-seq
33.3. Check the graph for how many cycles line up with this number from the curve.

In the case above it would be 6 because of where the line was
SECTION: ATAC-seq
34.1. # of addtional cycles

 

SECTION: ATAC-seq
36. Use the Zymo DNA Clean & Concentrator to clean the nuclei


 

SECTION: ATAC-seq
37. Quality control
TapeStation (DS1000) and Qubit quantification
SECTION: ATAC-seq
36.1. Add 2-7 volumes of the DNA binding buffer to each volume of DNA sample

So in this case the volume is approximately 50 ul, so add 300 ul of DNA binding buffer
SECTION: ATAC-seq
36.2. Transfer mixture to a provided Zymo-spin column in a collection tube
SECTION: ATAC-seq
36.3. Centrifuge 10000 x g, 30 s 
Discard supernatant
SECTION: ATAC-seq
36.4. Add 200 µL DNA wash buffer to the column

Centrifuge 10000 x g, 30 s 
SECTION: ATAC-seq
36.5. Repeat the wash step
SECTION: ATAC-seq
36.6. Transfer the column to a 1.5 low-bind Eppendorf tube.

Add 21 ul Sterile water buffer to the column.

Centrifuge 10000 x g, 30 s 
SECTION: Pre-experimentation
4.4. iii) ATAC-RSB: 
      		Amount 		Reagents 	  		500ul 		1M Tris-Hcl ph 7.5 	  		100ul 		5M NaCl 	  		150ul 		1M MgCl2 	  		49.25ml 		H2O 	 
 
SECTION: Pre-experimentation
4.5. iv)  ATAC-RSB + 10% Tween  
      		Amount 		Reagents 	  		8 ml 		ATAC-RSB 	  		8 ul 		10% Tween 	 
 

SECTION: Pre-experimentation
4.6. v) 2X TD buffer
 
      		Amount 		Reagents 	  		2 ml 		1 M Tris-Hcl pH 7.5 	  		1 ml 		1 M MgCl2 	  		20 ml 		100% Dimethyl Formamide 	 
 (before the addition of dimethyl formamide, adjust the pH to 7.6 with 100% acetic acid)

SECTION: Pre-experimentation
4.7. vi) 2X DF buffer

 
      		Amount 		Reagents 	  		100 mM 		HEPES (pH 7.2) 	  		200 mM 		NaCl 	  		0.2 mM 		EDTA 	  		2 mM 		DTT 	  		2.00% 		Triton X-100 	  		20.00% 		Glycerol 	 
 
SECTION: Nuclei extraction and filtration
15.1. Transfer the supernatant to a new tube without disturbing the pellet

Repeat the centrifugation