TITLE: S.O.F protocol for nuclei isolation from fresh and frozen tissues using OptiPrep® discontinuous gradient 
AUTHORS: Luciano Martelotto

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (S.O.F means Son Of Frankenstein). </div><div class = "text-block">Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® discontinuous gradient to remove debris. It is the alternative protocol when FACS is not available.</div></div>

[STEPS]
?. [Tissue Homogenization]
Mince/chop tissue with a razor blade to small pieces. The tissue may be as small as a grain of rice.
For mincing the tissue, you may take the tube out of ice, however, be quick and return to ice.
?. [Tissue Homogenization]
Add  of chilled Nuclei EZ Lysis Buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL) to the tissue in 1.5 mL tube.
300 µl
?. [Tissue Homogenization]
Gently homogenize the sample using a douncer by stroking 10-20 times. Keep tube on ice at all times.
?. [Nuclei Isolation]
Add an extra  of chilled Nuclei EZ Lysis Buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL), mix gently by pipetting using wide-bore tips and incubate on ice for . Repeat mixing 2-3 times during the incubation. RNAse inhibitor will now be in the range of 0.2-0.5 U/uL.
900 µl
?. [Nuclei Isolation]
Filter homogenate using a 70 μm-strainer mesh to fit a 15 ml Falcon tube (e.g. pluriStrainer Mini 70 μm Cell Strainer).
?. [Nuclei Isolation]
Transfer flow though into a 1.5 mL LoBind tube and centrifuge the nuclei at  for  at  and remove supernatant leaving behind ~.
4 °C
50 µl
?. [Nuclei Isolation]
Optional: add  of EZ Lysis buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL), gently resuspend pellet and incubate for  on ice. Then centrifuge the nuclei at  for  at .
1 mL
4 °C
?. [Nuclei Isolation]
Remove supernatant as much as possible without disturbing pellet (if pellet looks loose leave ~ behind) and add  of Nuclei Wash and Resuspension Buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL) without resuspending.
50 µl
1 mL
?. [Nuclei Isolation]
Centrifuge the nuclei at  for  at .
4 °C
?. [Nuclei Isolation]
Optional: Remove supernatant as much as possible without disturbing pellet (if pellet looks loose leave ~ behind) and add  of Nuclei Wash and Resuspension Buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL) without resuspending. Centrifuge the nuclei at  for  at .
50 µl
500 µl
4 °C
?. [Nuclei Isolation]
Remove supernatant as much as possible without disturbing pellet (if pellet looks loose leave ~ behind) and resuspend the pellet in  of G5 buffer and transfer onto a 10 mL LoBind centrifuge tube. Then add another mL of G5 (final volume 2 mL).
50 µl
1 mL
?. [Debris Removal]
Carefully, underlayer the sample with of the 30% iodixanol and then  of the 35% iodixanol, as shown in the picture below.
2 mL
2 mL
?. [Debris Removal]
Centrifuge the nuclei at  for  at  using swinging bucket centrifuge with break set to OFF. (Note: using is also possible).
4 °C
?. [Debris Removal]
After centrifugation, a white-ish band of nuclei between the 30%-35% iodixanol interface should be visible. Aspirate the top layers down until the white nuclei band at the interphase of 29%-35%. Using 200 uL tip, collect the nuclei band and transfer to a fresh 1.5 mL LoBind tube.
?. [Debris Removal]
Then top up to ~1.3 mL with Wash and Resuspension Buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL), mix well but gently, and centrifuge the nuclei at  for  at .
4 °C
?. [Debris Removal]
Remove supernatant and repeat once more for a total of 2 washes.
?. [Debris Removal]
Check integrity and purity under microscope and count manually or using an automatic counter. For automatic counter I recommend LUNA-FL™ Dual Fluorescence Cell Counter and Acridine Orange/Propidium Iodide (AO/PI) Cell Viability Kit (F23001).
?. [Debris Removal]
Proceed to your amazing snRNA-Seq experiment!