TITLE: Manual isolation of nuclei from human brain using CellRaft device and single nucleus Whole Genome Amplification
AUTHORS: Ester Kalef-Ezra, Diego Perez-Rodriguez, Christos Proukakis

[DESCRIPTION]
Protocol for manual nuclear isolation from human brain tissue using Cell Raft device for single cell Whole Genome Amplification.

[BEFORE_START]
Section 1: Nuclear extraction from human brain

Before starting:

1. Clean Human Tissue handling hood with 0.2 M NaOH, 10% Presept, 70% EtOH, and dH2O.
2. Clean Dounce tissue grinders (prior to use, between samples, and at the end of the experiment) with 0.2 M NaOH, 10% Presept, 70% EtOH, dH2O, and let them dry.
3. UV sterilize and clean PCR hood, centrifuge, pipettes, etc. with 10% Presept, 70% EtOH, and dH2O.
4. Precool centrifuge @4ºC.
5. On the day of the experiment prepare the following Buffers and keep themon ice:
 NIM (Nuclear Isolation Media): 25 mM KCl, 5 mM MgCl2, 10 mM Tris-HCl pH 8.8, 250 mM sucrose, 1 mM dithiothreitol (DTT), 1x cOmplete EDTA-free Protease Inhibitor Cocktail.
    ODN (Optiprep Diluent for Nuclei): 150 mM KCl, 30 mM MgCl2, 60 mM Tris-HCl pH 8.8, 250 mM sucrose, 1x cOmplete EDTA-free Protease Inhibitor Cocktail.
    25% iodixanol solution (NIM:ODGN:ODN 6:5:1).
    29% iodixanol solution (ODGN: ODN 29:31).
    NSB (Nuclei Storage Buffer) - if needed: 5 mM MgCl2, 50 mM Tris-HCl (pH 8.8), 166 mM sucrose and 1 mM dithiothreitol (DTT), 1x cOmplete EDTA-free Protease Inhibitor Cocktail.

C. Proceed to Immunodetection of inclusions and/or cell type marker staining

Before starting:

Prepare PBS: PBS 1x. Keep on ice until use.
Prepare PBS/PIC: PBS 1x supplemented with 1x cOmplete EDTA-free Protease Inhibitor Cocktail. Keep on ice until use.
Prepare Blocking Buffer: PBS+PIC supplemented with 10% goat serum. Keep on ice until use.

Section 3. View CytoSort Arrays under the microscope, capture & transfer rafts with single nuclei in tubes for single cell Whole Genome Amplification

Before starting: 

Defreeze an aliquot of diluted DNase I Solution and keep itat RT.
Prepare aliquots of 50 µl of 70% EtOH and 50 µl of dH2O in 1.5ml tubes in a PCR hood and keep at RT until use. You will need at least 1 aliquot of 70% EtOH and dH2O for each sample you want to amplify. As an example, if you aim to amplify the genome of 10 single nuclei, prepare 15 aliquots of 50 µl of 70% EtOH and 50 µl of dH2O. 
Prepare 0.2ml tubes with buffer depending on the downstream single cell Whole Genome Amplification (scWGA) method: (estimate double the number of tubes than the single nuclei to be collected):
           a. Multiple Displacement Amplification (MDA) in droplets (dMDA, Samplix): 2.8µl Lysis Bf (200 mM KOH, 5 mM   EDTA pH 8 and 40 mM 1.4 DTT) diluted in 2 µl dH2O
           b. PicoPlex (Takara): 5µl TE/Cell Extraction Buffer (depending on the version of the PicoPlex protocol used)
           c. Primary Template-directed Amplification/Resolve DNA (PTA, BioSkryB): 3µl Cell Buffer (BioSkryB)
Mount the CellRaft® Release Device onto a 4× or 10× objective of an inverted microscope and prepare the CytoSort Array for observation under the microscope. For details on this process please see the attached protocols (Attach CytoSort Array and CellRaft protocols):
              1. Cell Microsystems: CellRaft System for Inverted Microscopes (user manual)
              2. Cell Microsystems: CytoSort Array (user manual).
  

[GUIDELINES]
Critical note: Unless otherwise indicated, all the reagents must be kept at 4ºC and all the steps in the protocol must be performed at 4ºC. To preserve nuclear integrity, all the solutions are supplemented with complete cOmplete EDTA-free Protease Inhibitor Cocktail.


References:

Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development. 1992;116(1):201-211. doi:10.1242/dev.116.1.201.
Wolf HK, Buslei R, Schmidt-Kastner R, et al. NeuN: a useful neuronal marker for diagnostic histopathology. J Histochem Cytochem. 1996;44(10):1167-1171. doi:10.1177/44.10.8813082.
Cannon JR, Greenamyre JT. NeuN is not a reliable marker of dopamine neurons in rat substantia nigra. Neurosci Lett. 2009;464(1):14-17. 
Reed PJ, Wang M, Erwin JA et al (2017) Single-Cell Whole Genome Amplification and Sequencing to Study Neuronal Mosaicism and Diversity. In: Frade J., Gage F. (eds) Genomic Mosaicism in Neurons and Other Cell Types. Neuromethods, vol 131. Humana Press, New York, NY.

[STEPS]
SECTION: Nuclear extraction from human brain:
1. Transfer 20 mg -50 mg frozen brain tissue to microcentrifuge tube containing 500 µL of NIM. Transfer the tissue in a tube with the lid on using a pair of forceps.
SECTION: Nuclear extraction from human brain:
3. Gently triturate the tissue with a cut pipette tip (1 mL tip). Repeat several times with pipette tips cut less (estimate 3-4 different cut sizes).
 

SECTION: Nuclear extraction from human brain:
4. Measure the total volume using 1 mL pipette.
SECTION: Nuclear extraction from human brain:
5. Add Triton X-100 solution to the sample to achieve final concentration 0.1% (stock solution 10%).
SECTION: Nuclear extraction from human brain:
6. Homogenize using 2 mL Dounce tissue grinders:
a. Grinder A (loose, large clearance pestles): 2-3 strokes.
b. Grinder B (tight, small clearance pestles): 8-12 strokes.
 

SECTION: Nuclear extraction from human brain:
7. Transfer the homogenate to a fresh 1.5 mL microcentrifuge tube.
SECTION: Nuclear extraction from human brain:
8. Centrifuge at 1000 x g, 8 min, at 4 °C - Discard Supernatant.
SECTION: Nuclear extraction from human brain:
9. Resuspend the pellet gently  in 700 µL 25% Iodixanol Solution.
SECTION: Nuclear extraction from human brain:
10. Layer the sample (avoiding mixing the phases) onto 700 µL 29% iodixanol solution. Use 1 mL syringe without needle to slowly load and suspend the sample on top. As a result, 2 clearly defined phases should appear as follows:
  
SECTION: Nuclear extraction from human brain:
11. Spin at 10300 x g, 20 min, at 4 °C - remove the supernatant leaving 50 µL in the bottom of the ultracentrifuge tube (contain the nuclear fraction).
SECTION: Nuclear extraction from human brain:
12. At this point, follow any of the below routes:
SECTION: Nuclear extraction from human brain:
13. 
 

SECTION: Proceed to DAPI staining
13. Resuspend nuclei in DAPI (final 1 µg/ml) diluted in 1000 µL ice-cold PBS.

SECTION: Proceed to DAPI staining
14. Incubate for 20 min at 4 °C (or 60 min at 4 °C).
SECTION: Proceed to Immunodetection of inclusions
13. Immediately after isolation, resuspend the pelleted nuclei in 500 µL of pre-chilled Blocking buffer.
SECTION: Estimate nuclei number under the microscope:
14. Use a haemocytometer to estimate the number of nuclei in the final suspension. In a good nuclear fraction, most of the nuclei should be seen as single, with few clumps and/or low amount of cellular/tissue debris.
SECTION: Proceed to DAPI staining
15. Spin at 800 x g, for 10 min, at 4 °C - Remove the supernatant.
SECTION: Proceed to DAPI staining
16. Resuspend nuclear pellet in 500 µL ice-cold PBS.
SECTION: Proceed to DAPI staining
17. Check nuclei under the microscope using a hemacytometer.
SECTION: Estimate nuclei number under the microscope:
18. 
 

SECTION: Proceed to Immunodetection of inclusions
14. Incubate at 4 °C for 30 min with gentle shaking on a rotator disk to block non-specific binding sites.
SECTION: Proceed to Immunodetection of inclusions
15. Add primary antibody at the optimal concentration directly to the blocking buffer containing the nuclear suspension and incubate at 4 °C for 60 min with gentle shaking.
 

SECTION: Proceed to Immunodetection of inclusions
16. Pellet nuclei by centrifugation at 800 x g at 4 °C for 10 min, remove supernatant taking care not to disturb the pellet, and gently resuspend it in pre-chilled PBS to wash off excess primary antibody.
SECTION: Proceed to Immunodetection of inclusions
17. Pellet again the nuclei as stated above, discard supernatant.
SECTION: Proceed to Immunodetection of inclusions
18. Add 2 µg/ml goat anti-mouse and/or anti-rabbit secondary antibody and 1 µg/ml DAPI diluted in Blocking buffer.

SECTION: Proceed to Immunodetection of inclusions
19. Incubate for 60 min at 4 °C with gentle shaking on a rotator disk.
SECTION: Proceed to Immunodetection of inclusions
20. After incubation, wash the excess of PBS by pelleting again the nuclei and resuspending them in fresh PBS/PIC.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope:
15. 
 

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
16. Visually inspect the CytoSort array prior opening. The pre-sterilized arrays are expected to have a red sticker on packaging. Remove the array from the sealed pouch.  

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
18. Wash with 2 mL dH2O – incubate for 3 min at Room temperature – Remove water.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
19. Repeat the washing with 2 mL dH2O – incubate for 3 min at Room temperature - Remove water.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
20. Apply 1 mL of 15 ug/mL Cell-Tak in PBS with 2.5 millimolar (mM) NaOH – incubate for 20 min @Room temperature – Remove Cell-Talk.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
21. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
22. Repeat step 21 (3 times in total):

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
23. 
Optional step: Insert the CellRaft seeding insert to increase the overall number of single cells available for isolation.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
24. Dilute approximately 5000 nuclei in 2 mL of PBS, add pipette them dropwise in the centre of the CytoSort Array.
 

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
25. Move the CytoSort Array carefully to ensure the nuclei settle evenly within the array (like seeding cells for cell culture).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
26. Allow it to settle for 120 min  in the fridge.
 


SECTION: Isolation of rafts containing the nuclei of interest:
27. View CytoSort Arrays under the 10x magnification objective with appropriate fluorescence filters, to select rafts (microwells) containing a single nucleus with the desired characteristics and take photos (if needed Fig 4).
  

SECTION: Isolation of rafts containing the nuclei of interest:
28. Individually release the arrays of interest (Figure 5).
   

SECTION: Isolation of rafts containing the nuclei of interest:
29. Place the magnetic wand in the tubes containing DNase-I diluted solution.
 

SECTION: Isolation of rafts containing the nuclei of interest:
30. Wash the wand with 70% EtOH and then with dH2O.
SECTION: Isolation of rafts containing the nuclei of interest:
31. Capture, release and remove the released raft to the already prepared 0.2 mL tubes with the buffer needed for scWGA using the magnetic want. Then, centrifuge the tubes briefly.
SECTION: Isolation of rafts containing the nuclei of interest:
32. When performing CellRaft isolation, it is highly recommended to ensure the rafts are properly released at the bottom of the tube and not in the tube wall, as they would desiccate. To do so, visually inspect every tube after collection under a stereoscopic microscope; rafts will appear as small orange squares.
 


  

SECTION: Isolation of rafts containing the nuclei of interest:
33. Freeze the samples at -80 °C or proceed directly to scWGA.

For scWGA we have used the following methodologies:

PicoPlex (R300670, R300672 and R300722) according to Takara’s protocols.
dMDA with heat denaturation and Buffer Generation (dMDA protocol attached).
PTA (PTA protocol attached) but for step 7: incubate for 20 min at Room temperature.
 

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope:
19. 
 

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
20. Visually inspect the CytoSort array prior opening. The pre-sterilized arrays are expected to have a red sticker on packaging. Remove the array from the sealed pouch.

  

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
22. Wash with 2 mL dH2O – incubate for 3 min at Room temperature – Remove water.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
23. Repeat the washing with 2 mL dH2O – incubate for 3 min at Room temperature - Remove water.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
24. Apply 1 mL of 15 µg/ml Cell-Tak in PBS with 2.5 millimolar (mM) NaOH – incubate for 20 min at Room temperature – Remove Cell-Talk.

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
25. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
26. Repeat step 25 (3 times in total).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
27. 

Optional step: Insert the CellRaft seeding insert to increase the overall number of single cells available for isolation.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
28. Dilute approximately 5000 nuclei in 2 mL of PBS, add pipette them dropwise in the centre of the CytoSort Array.
 

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
29. Move carefully the CytoSort Array to ensure the nuclei settle evenly within the array (like seeding cells for cell culture).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
30. Allow it to settle for 120 min  in the fridge.
 

SECTION: Isolation of rafts containing the nuclei of interest:
31. View CytoSort Arrays under the 10x magnification objective with appropriate fluorescence filters, to select rafts (microwells) containing a single nucleus with the desired characteristics and take photos (if needed) (Fig 4)
   

SECTION: Isolation of rafts containing the nuclei of interest:
32. Individually release the arrays of interest (Figure 5).
   

SECTION: Isolation of rafts containing the nuclei of interest:
33. Place the magnetic wand in the tubes containing DNase-I diluted solution.
 

SECTION: Isolation of rafts containing the nuclei of interest:
34. Wash the wand with 70% EtOH and then with dH2O.
SECTION: Isolation of rafts containing the nuclei of interest:
35. Capture, release and remove the released raft to the already prepared 0.2 mL tubes with the buffer needed for scWGA using the magnetic want. Then, centrifuge the tubes briefly (Figure 6).
 
  
SECTION: Isolation of rafts containing the nuclei of interest:
36. When performing CellRaft isolation, it is highly recommended to ensure the rafts are properly released at the bottom of the tube and not in the tube wall, as they would desiccate. To do so, visually inspect every tube after collection under a stereoscopic microscope; rafts will appear as small orange squares.
 

SECTION: Isolation of rafts containing the nuclei of interest:
37. Freeze the samples at -80 °C or proceed directly to scWGA.
For scWGA we have used the following methodologies:
PicoPlex (R300670, R300672 and R300722) according to Takara’s protocols.
dMDA with heat denaturation and Buffer Generation (dMDA protocol attached).
PTA (PTA protocol attached) but for step 7: incubate for 20 min at Room temperature.
 

SECTION: Estimate nuclei number under the microscope:
21. 
 

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope:
22. 
 

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
23. Visually inspect the CytoSort array prior opening. The pre-sterilized arrays are expected to have a red sticker on packaging. Remove the array from the sealed pouch.
  
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
25. Wash with 2 mL dH2O – incubate for 3 min at Room temperature – Remove water.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
26. Repeat the washing with 2 mL dH2O – incubate for 3 min at Room temperature - Remove water.

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
27. Apply 1 mL of 15 µg/ml Cell-Tak in PBS with 2.5 millimolar (mM) NaOH – incubate for 20 min atRoom temperature – Remove Cell-Talk.

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
28. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
29. Repeat step 28 (3 times in total).
Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS (1/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
30. 

Optional step: Insert the CellRaft seeding insert to increase the overall number of single cells available for isolation.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
31. Dilute approximately 5000 nuclei in 2 mL of PBS, add pipette them dropwise in the centre of the CytoSort Array.
 


SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
32. Move carefully the CytoSort Array to ensure the nuclei settle evenly within the array (like seeding cells for cell culture).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-Seeding nuclei to the CytoSort array:
33. Allow it to settle for 120 min  in the fridge.
 

SECTION: Isolation of rafts containing the nuclei of interest:
34. View CytoSort Arrays under the 10x magnification objective with appropriate fluorescence filters, to select rafts (microwells) containing a single nucleus with the desired characteristics and take photos (if needed) (Fig 4).

   

SECTION: Isolation of rafts containing the nuclei of interest:
35. Individually release the arrays of interest (Figure 5).

   

SECTION: Isolation of rafts containing the nuclei of interest:
36. Place the magnetic wand in the tubes containing DNase-I diluted solution.
 

SECTION: Isolation of rafts containing the nuclei of interest:
37. Wash the wand with 70% EtOH and then with dH2O.
SECTION: Isolation of rafts containing the nuclei of interest:
38. Capture, release and remove the released raft to the already prepared 0.2 mL tubes with the buffer needed for scWGA using the magnetic want. Then, centrifuge the tubes briefly  (Figure 6).
   

SECTION: Isolation of rafts containing the nuclei of interest:
39. When performing CellRaft isolation, it is highly recommended to ensure the rafts are properly released at the bottom of the tube and not in the tube wall, as they would desiccate. To do so, visually inspect every tube after collection under a stereoscopic microscope; rafts will appear as small orange squares.
 

SECTION: Isolation of rafts containing the nuclei of interest:
40. Freeze the samples at -80 °C or proceed directly to scWGA.

For scWGA we have used the following methodologies:

PicoPlex (R300670, R300672 and R300722) according to Takara’s protocols
dMDA with heat denaturation and Buffer Generation (dMDA protocol attached).
PTA (PTA protocol attached) but for step 7: incubate for 20 min at Room temperature.
 

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
29.1. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS (2/3)
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
29.2. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS (3/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
26.1. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS(1/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
26.3. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS(3/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
26.2. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS(2/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
22.1. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS (1/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
22.3. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS (3/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
22.2. Add 2 mL sterile PBS 1x - Incubate for 3 min at Room temperature – Remove PBS (2/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
17. Optional step: It may be possible to observe crystallization of excess glucose that was used to seal the CytoSort array from exposed air (Figure 2B). In that case:
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
17.1. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
17.6. Continue to the next steps.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
17.5. Cool the array at Room temperature .

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
17.2. Repeat above step 3 times in total:

Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min (1/3).

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
17.3. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min (2/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
17.4. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min (3/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
21. Optional step: It may be possible to observe crystallization of excess glucose that was used to seal the CytoSort array from exposed air (Figure 2B). In that case:

SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
21.1. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
21.2. Repeat above step 3 times in total.
Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min (1/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
21.5. Cool the array at Room temperature.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
21.6. Continue to the next steps.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
21.4. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min (3/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
21.3. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min (2/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
24. Optional step: It may be possible to observe crystallization of excess glucose that was used to seal the CytoSort array from exposed air (Figure 2B). In that case:
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
24.1. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
24.6. Continue to the next steps.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
24.2. Repeat above step 3 times in total.
Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min(1/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
24.5. Cool the array at Room temperature.
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
24.4. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min(3/3).
SECTION: Manual isolation of single nuclei using a device fixed to an inverted microscope-CytoSort Array Preparation:
24.3. Wash the array with 2 mL (pre-warmed at 37 °C) PBS 1x – incubate for 3 min(2/3).
SECTION: Nuclear extraction from human brain:
2. Optional step: Mincing the tissue in small fragments over a glass plate on ice using a scalpel could significantly decrease the time needed for homogenization and facilitate the trituration with the pipette tips.