Single cell ATAC-seq

1. Sacrifice mice via decapitation according to institutional guidelines

2. Dissect out the segment of interest ot the spinal column and directly place on ice-cold dissection buffer

3. Transfer the tissue to a petri dish containing ice-cold dissection buffer placed under a dissection microscope and microdissect the segment of interest of the spinal cord removing meningeal coverage.

4. Transfer the dissected tissue onto a C Tube (Miltenyi) containing the appropriate volume of pre-warmed Buffer X and enzyme P (Neural Tissue Dissociation Kit (P), Miltenyi) according to the manufacturer’s instructions

5. Place the C Tube on a Gentle MACS dissociator (Miltenyi) and run program m_brain_01.

6. Incubate for 15 minutes at 37C with slow rotation to digest the minced tissue.

7. Place the C Tube on a Gentle MACS dissociator (Miltenyi) and run program m_brain_02.

8. Add 30 μl of the mix of Buffer Y and Enzyme A according to the manufacturer’s instructions.

9. Incubate for 10 minutes at 37C with slow rotation. The suspension will start to appear milky, with some tissue pieces.

10. Place the C Tube on a Gentle MACS dissociator (Miltenyi) and run program m_brain_03.

11. Incubate for 10 minutes at 37C with slow rotation. The suspension will now appear almost completely milky.

12. Centrifuge for 10’’ at 500G to collect the suspension to the bottom of the tube

13. Add 15 ml of ice-cold  flow cytometry buffer (HBSS buffer supplemented with 10% fetal calf serum) to stop the digestion and pass the cell suspension through a 70 μm strainer

14. Collect the cells by centrifugation at 500 G for 10’ at 4C

15. Resuspend in an appropriate volume of flow cytometry buffer and proceed to myelin removal with Myelin Removal Beads (Milteny) according to the manufacturer’s instructions

16. Add the appropriate volume of myelin removal beads and incubate for 15 minutes on ice. Antibody staining may be performed at this step

17. Wash the cells twice in 2 ml of flow cytometry buffer

18. Pass the cell suspension through an LS column and collect the flow trhough fractions

19. Collect the cells by centrifugation for 5’ at 500 G at 4C

20. Resuspend the cells in flow cytometry buffer and sort desired population in a flow cytometer

21. Collect cells in flow cytometry buffer

22. Lyse cells according to protocol CG000212 (10X Genomics) by adding 100 μl of 0.1X Lysis Buffer and pipette up and down 5 times

23. Incubate for 5 minutes on ice

24. Add 1ml of cold Wash Buffer to the lysed cells and pipette up and down 5 times gently

25. Collect the nuclei by centrifugation for 5’ at 500 G at 4C

26. Slowly remove most of the supernatant without disrupting the nuclei pellet

27.  Add 100ul of Diluted Nuclei buffer but do not resuspend

28. Centrifuge for 3’ at 500 G at 4C

29. Slowly remove the supernatant until there is approximately 10 μl left (volume will depend on the number of cells sorted)

30. Take 1 μl of the resuspended cells in diluted nuclei buffer to determine the number of intact nuclei in a counting chamber (dilute 1:10 in DPBS containing trypan blue)

31. Transfer the volume corresponding to the desired number of nuclei according to Chromium Next GEM Single Cell ATAC Reagent kits v1.1 (10X Genomics; CG000209) to a 0,2 ml PCR tube.

32. Proceed exactly as specified in 10X Genomics user guide CG000209 Rev D.

Dissection buffer (sterile-filtered):


HBSS (without Ca2+ and Mg2+) 1X


Penicilin-Streptomicin 100 U/ml


HEPES 10 mM


D-Glucose 0,81% w/v

0.1X Lysis buffer:

Tris-HCL (pH 7.4)          1mM

NaCl 1mM

MgCl2         0.3mM

Tween-20    0.01%

NP-40         0.01%

Digitonin     0.001%

BSA   0.1%

Wash buffer:

Tris-HCL (pH 7.4)          10mM

NaCl 10mM

MgCl2         3mM

Tween-20    0.1%

BSA   1%

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