TITLE: Isolation of single nuclei from postmortem fresh frozen human brain and immunostaining for NeuN
AUTHORS: Marcos Otero-Garcia, Inma Cobos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>- Protocol based on Krishnaswami </span><span style = "font-style:italic;">et al.</span><span>, Nat Protoc. 2016, 3:499-524</span></div><div class = "text-block">- Used routinely in our lab for the isolation of single nuclei from fresh frozen postmortem brains from subjects with neurodegenerative disorders and healthy controls for single-nucleus RNA-seq</div><div class = "text-block">- Works also for isolation of nuclei from fresh frozen mouse brains </div><div class = "text-block"><span>- Used for isolation of all nuclei or antibody-enriched populations (i.e., NeuN</span><span style = "vertical-align:super;">+</span><span> nuclei)</span></div><div class = "text-block">- We always perform and strongly recommend FACS if used for single-nucleus RNA-seq</div></div>

[STEPS]
?. Prepare Homogenization Buffer and cool it on ice.   ABC1AmountReagentFinal concentration22925 µlIM133 µlDTT 1mM1 µM430 µl50x Protease Inhibitor0.5 x515 µlRNaseIN 40U/µl0.2 U/µl630 µlTritonX100 10 %v/v0.1 %
ABC1AmountReagentFinal concentration22925 µlIM133 µlDTT 1mM1 µM430 µl50x Protease Inhibitor0.5 x515 µlRNaseIN 40U/µl0.2 U/µl630 µlTritonX100 10 %v/v0.1 %
?. Pre-cool the dounce tissue grinder on ice  (Kimble Kontes all glass tissue grinder, 7 mL tubes, 0.02 - 0.056 mm clearance space between pestle and tubes)
?. Add  of Homogenization buffer to the dounce tissue grinder.
2.4 ml
?. Collect the brain chunk (~) and transfer to a Petri dish on ice.
100 mg
?. Cut out into small pieces using a chilled scalpel or blade.
?. Transfer all pieces into the dounce tissue grinder.
?. Homogenize the tissue, on ice (~30 firm strokes). Check on hematocytometer while homogenizing and adjust the number of strokes.
Too few strokes – you will see cell clumps; too many strokes – you will get damaged nuclei.
?. Filter homogenate using 40 µm Corning cell strainer to remove clumps.--> Take sample for hemocytometer.
?. Transfer the homogenate into two precooled 1.5 ml Eppendorf tubes.
?. Centrifuge at  for  at .
4 °C
?. Slowly aspirate the supernatant from the side of the tube. Avoid disturbing the pellets, up to  of supernatant can be left.
50 µl
?. Gently resuspend each pellet in  (final volume) of cold Homogenization buffer and pool both tubes (final volume ).
225 µl
450 µl
?. Add an equal volume of cold  .
[iodixanol]
Critical! Be very exact with volumes.   suspension +   50 % iod. Final iodixanol concentration is 25 %.
?. Gently pipette mix.
?. Add an equal volume of 29 % iodixanol () into a 2 ml Eppendorf tube.
900 µl
?. Slowly layer off the 25 % iodixanol suspension mix over the 25 % iodixanol, without mixing them.
?. Centrifuge at  for  at .
4 °C
?. Prepare buffer for immunostaining.  ABC1AmountReagentFinal concentration29400 µlRNase-free PBS (pH7.4)3500 µlRNase-free 10 % BSA0.5 %450 µl1 M MgCl25 mM510 µlDNAse I (2000 U/ml)2 U/ml
ABC1AmountReagentFinal concentration29400 µlRNase-free PBS (pH7.4)3500 µlRNase-free 10 % BSA0.5 %450 µl1 M MgCl25 mM510 µlDNAse I (2000 U/ml)2 U/ml
?. After centrifugation, remove and discard the top myelin-rich debris layer (you can use a 1 ml pipette with the tip cut, or cotton swabs).
?. Then, remove and discard the aqueous supernatant, without disrupting the nuclei pellet. Avoid contaminating with the top layer.
?. Use a small amount of buffer for Antibody Incubation to resuspend the pellet and transfer the solution to a new tube.
?. Gently resuspend in  of buffer for Antibody Incubation.
500 µl
?. Incubate for , at  or iced, for blocking nonspecific staining.
4 °C
?. Take sample for hemocytometer.
?. Take a sample for unstained control. Take sample for 2AB-only (nonspecific binding control).
?. Add primary antibody (Ms-a-NeuN, 1:1000) and incubate on a rotator in cold room () for .
4 °C
Use Eppendorfs coated with BSA for collection (to coat the Eppendorf tubes, fill them with 10 % BSA solution in PBS for  , rinse with PBS, and dry at   overnight).
?. [Washing]
Add  of buffer for Immunostaining and invert several times.
700 µl
?. [Washing]
Centrifuge at  for  at .
4 °C
?. [Washing]
Carefully transfer all the supernatant (don’t leave any supernatant! It’s ok to drag some pellet) to a new Eppendorf tube and resuspend the pellet in  of buffer for Antibody Incubation.
400 µl
?. [Washing]
Centrifuge the supernatant at  for  at  to recover non-pelleted nuclei.
4 °C
?. [Washing]
Resuspend them in  of buffer for Antibody Incubation and pool for a final volume of 600 µl.
200 µl
?. Take samples for single color staining.
?. Add secondary antibody (G-a-Ms Alexa Fluor 647, 1:1000) and Hoechst 33258 (5 µg/ml) to the same tube. (Hoechst 1:1000 of 2,5 mg/ml stock).
?. Check the quality of the sample and on hemocytometer.
For 100 mg of tissue, should have ~ 2 Million nuclei in ~0.6 ml.
?. Transfer to 7 ml culture tubes.
?. Place on ice and bring them to the FACS facility for sorting.
FACS sorting controls:Non-stained2AB onlySingle stainings
?. Collect in BSA coated Eppendorfs containing collection medium (containing ~1/5 of the expected final volume after collection).
?. Add BSA after collection for final 1% BSA final concentration (tested for 10x Chromium v2 and v3).
Different Single Cell assays tolerate different BSA concentrations, but lowering it may increase nuclei aggregation.
?. Add .
[IM1]
?. Add .
[DTT 1mM]
?. Add .
[50x Protease Inhibitor]
?. Add .
[RNaseIN 40U/µl]
?. Add .
[TritonX100 10 %v/v]
?. Add .
[RNase-free PBS (pH7.4)]
?. Add .
[RNase-free 10 % BSA]
?. Add .
[1 M MgCl2]
?. Add .
[DNAse I (2000 U/ml)]
?. Prepare buffer for Antibody incubation (1.5 ml per sample) by adding   of RNaseIN 40 U/µl to  of buffer for Immunostaining (final concentration 0.2U/µl)
5 µl
1 ml