TITLE: SPLiT-seq Nuclei isolation for Micro-dissected Mouse Brain Tissue 
AUTHORS: Ashley B Robbins

[DESCRIPTION]
Protocol for nuclei isolation prior to SPLiT-seq library preparation. Specifically optimized for the storage of nuclei from micro-dissected brain regions from mice or human (50-100mg). 

[GUIDELINES]
Tissue collection for this protocol requires prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.

[STEPS]
SECTION: Setup
2. Prepare stock lysis buffer (can be stored at 4°C for 1 week)

For 15 mL stock:


1.641 g Sucrose
75 µL  1M CaCl2
45 µL 1M Mg(Ac)2
3 µL 0.5M EDTA
150 µL Tris-HCl, pH 8.0
15 µL Triton X-100

Fill to 15 mL  with UltraPure H2O (DNase-free, RNase-free)

SECTION: Setup
3. Prepare wash & resuspension buffer

For 12 mL:

1.2 mL Bovine Serum Albumin Fraction V, 7.5% solution
120 µL SUPERase•In™ RNase Inhibitor, 20 U/uL
120 µL RNaseOUT™ Recombinant Ribonuclease Inhibitor, 5,000 units

Fill to 12 mL  with 1X PBS

SECTION: Dounce Homogenization
4. Add DTT and RNase inhibitors to lysis buffer

Aliquot 1 mL of stock lysis buffer per sample (~100 mg)

Per 1 mL (per sample)

Add 1:1000 1,4-Dithiothreitol (DTT)
Add 5 µL  SUPERase•In™ RNase Inhibitor, 20 U/uL per mL
Add 5 µL  RNaseOUT™ Recombinant Ribonuclease Inhibitor, 5,000 units

SECTION: Dounce Homogenization
5. Add 500 µL of lysis buffer (+ DTT & RNase inhibitors) to each sample (in a 1.5 mL eppendorf tube or cryovial)

SECTION: Dounce Homogenization
6. Incubate for 10 minutes on ice. 

 Incubation time may need to be optimized depending on tissue 
SECTION: Dounce Homogenization
7. With a wide-pore P1000 barrier pipet tip homogenize the tissue by pipetting up and down (3-5X) until the tissue readily passes through the pipet tip.
SECTION: Dounce Homogenization
8. Transfer tissue homogenate to the dounce homogenizer with a wide-pore P1000 barrier pipet tip.
SECTION: Dounce Homogenization
9. Wash the sample tube with of lysis buffer (+ DTT & RNase inhibitors) and transfer buffer to the dounce homogenizer.

 This step ensures optimal retention of semi-homogenized tissue for dounce homogenization
SECTION: Dounce Homogenization
10. Dounce 10-30 times with Pestle A 

 The # of dounces should be optimized by tissue
SECTION: Washing
12. Pass 500 µL at a time through a 40 µM strainer into a 1.5 mL LoBind Eppendorf tube (pre-chilled) using a wide-pore P1000 barrier pipet tip. 
 

 

SECTION: Washing
13. Centrifuge at 600 x g for 10 min. @ 4°C. 

 The spin speed can be adjusted between 300 - 800 x g, faster spins risk damage to the nuclear membrane. 
SECTION: Dounce Homogenization
11. Dounce 5-10 times with Pestle B

 The # of dounces should be optimized by tissue
SECTION: Fixation and Permeabilization
23. Prepare solutions for fixation & permeabilization and place on ice:

Fixation Solution:

750 µL 16% Formaldehyde
2.25 mL 1X PBS

Permeabilization Solution:

2 mL 5% Triton X-100 
2 µL SUPERase•In™ RNase Inhibitor, 20 U/uL

Neutralization Buffer:

500 µL 1M Tris-HCl, pH 8.0
200 µL 5% Triton X-100 
4.8 mL 1X PBS

SECTION: Fixation and Permeabilization
24. Filter nuclei through a 40 µM strainer into a 1.5 mL LoBind Eppendorf tube (pre-chilled) using a wide-pore P1000 barrier pipet tip to a 15 mL conical tube. 


 

SECTION: Nuclei Storage
32. Remove the supernatant and add 300 µL of Nuclei Storage Buffer. Let the pellet sit for 2 min. on ice prior to re-suspending.

SECTION: Nuclei Storage
31. Prepare nuclei storage buffer and place on ice:

Nuclei Storage Buffer:

6 mL 1X PBS
15 µL SUPERase•In™ RNase Inhibitor, 20 U/uL
7.5 µL RNaseOUT™ Recombinant Ribonuclease Inhibitor, 5,000 units

SECTION: Washing
17. Repeat steps 14-16.   

 

SECTION: Washing
14. Remove the supernatant and add 500 µL of wash & resuspension buffer. Let the pellet sit for 2 min. on ice prior to re-suspending.

SECTION: Washing
15. Gently re-suspend the nuclei pellet with a wide-pore P1000 barrier pipet tip. 

SECTION: Washing
16. Centrifuge at 600 x g for 5 min. @ 4°C. 
SECTION: Washing
18. Remove the supernatant and add 500 µL of wash & resuspension buffer. Let the pellet sit for 2 min. on ice prior to re-suspending.


SECTION: Washing
19. Gently re-suspend the nuclei pellet with a wide-pore P1000 barrier pipet tip. 
SECTION: Washing
20. Pass the resuspended nuclei through a 40 µm Flow cell strainer into a 1.5 mL LoBind Eppendorf tube (pre-chilled).
SECTION: Washing
21. Centrifuge at 600 x g for 5 min. @ 4°C. 
SECTION: Washing
22. Remove the supernatant and add 750 µL of wash & resuspension buffer. Let the pellet sit for 2 min. on ice prior to re-suspending.


 

SECTION: Fixation and Permeabilization
25. Add 250 µL of pre-chilled Fixation Solution and immediately mix gently by pipetting up and down 3X with a wide-pore P1000 barrier pipet tip. 


 



SECTION: Fixation and Permeabilization
26. Incubate for 10 minutes on ice. 
SECTION: Fixation and Permeabilization
27. Add 40 µL of Permeabilization Solution and mix gently by pipetting up and down 3X with a wide-pore P1000 barrier pipet tip. 

SECTION: Fixation and Permeabilization
28. Incubate for 3 minutes on ice. 
30. Centrifuge at 500 x g for 5 min. @ 4°C in the swinging bucket rotator.
SECTION: Nuclei Storage
33. Gently re-suspend the nuclei pellet with a wide-pore P1000 barrier pipet tip. 
SECTION: Nuclei Storage
34. Filter nuclei through a 40 µM strainer into a 1.5 mL LoBind Eppendorf tube (pre-chilled) using a wide-pore P1000 barrier pipet tip. 
SECTION: Nuclei Storage
35. Add 5 µL of DMSO and gently flick the tube 3X, wait 1 minute on ice. Repeat 3X to add a total of 15 µL DMSO. 

SECTION: Nuclei Storage
36. Mix the final suspension 5X with a P200 barrier pipet tip and aliquot for future experiments.
SECTION: Nuclei Storage
37. Place aliquoted nuclei suspensions in a Mr. Frosty to cool at -1°C/minute in a -80°C freezer overnight. 
SECTION: Nuclei Storage
38. Fixed nuclei suspensions can be stored at -80°C for up to 6 months. 
SECTION: Fixation and Permeabilization
29. Add 1 mL cold nuclei neutralization buffer and gently invert the tube 3X to mix
SECTION: Setup
1. Chill dounce homogenizers and tubes at 4°C in wet ice bucket

Per sample:

2mL x 60 mm KIMBLE Dounce tissue grinder set
(2) DNA LoBind® Tubes, 1.5 mL