TITLE:  ‘Frankenstein’ protocol for nuclei isolation from FRESH and FROZEN tissue for snRNA-Seq (10x genomics Platform)
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, hence the name Frankenstein. Developed to prepare nuclei isolates from small sample sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al.</div><div class = "text-block">This protocol has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide for single nuclei experiments using 10x Genomics technologies.</div><div class = "text-block">The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and other samples types.</div><div class = "text-block"><span style = "font-weight:bold;">Cardiomyocytes </span><span>can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.  </span></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  chilled Nuclei EZ Lysis Buffer to the tissue in 1.5 mL tube.
500 µl
?. [Tissue Homogenization]
Homogenize the sample using a douncer (stroking 10-20 times).
?. [Tissue Homogenization]
Transfer the homogenate (~) into a 2 mL tube.
500 µl
?. [Nuclei Isolation and Staining]
Add  of chilled Nuclei EZ Lysis Buffer, mix gently and incubate on ice for . Gently mix with a wide bore tip.  Repeat 1-2 times during the incubation.
1 mL
?. [Nuclei Isolation and Staining]
Filter homogenate using a 70 μm-strainer mesh. Collect flow through in a polystyrene round-bottom FACS tube and transfer volume back into a new 2 mL tube.
?. [Nuclei Isolation and Staining]
Centrifuge the nuclei at  for  at  and remove supernatant leaving behind ~.
4 °C
50 µl
?. [Nuclei Isolation and Staining]
Gently resuspend nuclei in another  of EZ Lysis buffer, incubate for  on ice.
1.5 mL
?. [Nuclei Isolation and Staining]
Centrifuge the nuclei at  for  at , remove supernatant as much as possible without disturbing pellet (if pellet looks loose leave ~ behind).
4 °C
50 µl
?. [Nuclei Isolation and Staining]
Add  Nuclei Wash and Resuspension Buffer and incubate  without resuspending to allow buffer interchange.
500 µl
?. [Nuclei Isolation and Staining]
After incubation, add  of Nuclei Wash and Resuspension Buffer and resuspend the nuclei.
1 mL
?. [Nuclei Isolation and Staining]
Centrifuge the nuclei at  for  at , remove supernatant leaving behind ~.
4 °C
50 µl
?. [Nuclei Isolation and Staining]
Gently resuspend nuclei in  Nuclei Wash and Resuspension Buffer and transfer to a 1.5 mL tube (easier to see small pellets).
1.4 mL
?. [Nuclei Isolation and Staining]
Repeat Step 13 and resuspend in  Nuclei Wash and Resuspension Buffer supplemented with DAPI. Collect all nuclei by washing off nuclei from the wall of centrifuge tube.
500 µl
IMPORTANT: Protect from light from here forward.
?. [Cytometry and 10x  ]
Filter nuclei (at least once) with a 35-µm cell strainer.  Visually inspect nuclei integrity under a microscope.
Count the number of nuclei with a cell counter or hematocytometer if required.
?. [Cytometry and 10x  ]
Perform cytometric analysis. Identify single nuclei and sub-populations based on DNA content, gate and sort directly into 10x RT Buffer prepared without the RT Enzyme Mix.
?. [Cytometry and 10x  ]
Proceed immediately with the 10x Genomics Single Cell Protocol and minimize the time between nuclei preparation/sorting and chip loading.
?. [Cytometry and 10x  ]
Add  RT Enzyme Mix to the sorted nuclei in RT buffer, mix well but gently and load chip as per the  Single Cell 3' Reagents User Guide or Single Cell V(D)J 5' Reagents User Guide.
10 µl
?. [Cytometry and 10x  ]