TITLE: Daughter of Frankenstein protocol for nuclei isolation from fresh and frozen tissues using OptiPrep® continuous 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 (D.O.F means Daughter Of Frankenstein). </div><div class = "text-block"><span>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® </span><span style = "font-weight:bold;font-style:italic;">continuous</span><span> gradient to remove debris. It is the alternative protocol when FACS is not available. </span></div><div class = "text-block">This protocol works better with smaller samples. After centrifugation the nuclei will form a pellet and contaminating membranes will float at the top. This continuous gradient can be applied for as many times as needed to the same prep to continue cleaning up your nuclei suspensions. </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 it 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 nuclei suspension on ice at all times.
?. [Tissue Homogenization]
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.
700 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 (optional: incubate for  on ice for additional lysis). 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 G30 buffer. Mix well but gently to resuspend the pellet.
50 µl
300 µl
?. [Nuclei Isolation]
Carefully, underlay the sample with of the G30. A clear separation between homogenate and added G30 will form. Do not disturb.
1 mL
?. [Nuclei Isolation]
Centrifuge the nuclei at  for  at . After centrifugation the nuclei will form a pellet and contaminating membranes will float at the top. This continuous gradient can be applied for as many times as needed to the same prep to continue cleaning up your nuclei suspensions (Optional Step 11).
Centrifuge: 8000 34
4 °C
?. [Nuclei Isolation]
Optional: repeat steps 8-10) for additional cleaning.
?. [Nuclei Wash and Resuspension]
After centrifugation, remove supernatant and add  of ice-cold Nuclei Wash and Resuspension Buffer supplemented (supplemented with RNAse Inhibitor 0.2-0.5 U/uL) without resuspending the nuclei. Then centrifuge the nuclei at  for  at .
1 mL
4 °C
?. [Nuclei Wash and Resuspension]
Remove supernatant as much as possible without disturbing pellet (if pellet looks loose leave ~ behind) and resuspend the pellet in  of ice-cold Nuclei Wash and Resuspension Buffer supplemented (supplemented with RNAse Inhibitor 0.2-0.5 U/uL). Then centrifuge the nuclei at  for  at .
50 µl
1 mL
4 °C
?. [Nuclei Wash and Resuspension]
Remove supernatant and resuspend with  of ice-cold Nuclei Wash and Resuspension Buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL) and filter using 40-um Flowmi® Cell Strainer and transfer to a new LoBind 1.5 mL Eppendorf tube.
300 µl
?. [Nuclei Wash and Resuspension]
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).
?. [Nuclei Wash and Resuspension]
Proceed to your amazing snRNA-Seq experiment!