TITLE: TST Nuclei Isolation with GentleMACS - 220301
AUTHORS: Sébastien Vigneau

[DESCRIPTION]
This protocol describes the process of nuclei isolation from frozen tissue. The protocol has been applied to frozen melanoma, breast, and lung metastases for the Human Tumor Atlas Network (HTAN) single-nuclei RNA-seq preparation.

[STEPS]
1. Buffer Preparation

Prepare the necessary buffers and solutions as outlined below:

2x ST (50 mL stock solution can be prepared ahead of time and stored at room temperature)
  

TST (2 mL should be prepared for each tissue sample)
*10% Tween-20 can be prepared ahead of time and stored at 4ºC
  

Reagent


Volume for 2 mL Working Solution (per Sample)

 

2x ST stock solution


1000 μL

 

MACS BSA Stock Solution (10%)


2 μL (0.01% final concentration)

 

10% Tween-20*


6 μL

 

H2O


942 μL

 

Protector RNase Inhibitor (40 U/μL)


50 μL (1 U/μL final concentration)

  

1x ST (3.5 mL should be prepared for each tissue sample)
  

Reagent


Volume for 3.5 mL Working Solution (per Sample)

 

2x ST stock solution


1748 μL

 

H2O


1664.5 μL

 

Protector RNase Inhibitor (40 U/μL)


87.5 μL

  

PBS + BSA (1%) + RNase Inhibitors (1 mL should be prepared for each tissue sample)
  

Reagent


Volume for 3.5 mL Working Solution (per Sample)

 

PBS 1X


875 μL

 

MACS BSA Stock Solution (10%)


100 μL

 

Protector RNase Inhibitor (40 U/μL)


25 μL

  

2. Tissue Dissociation

Fill a GentleMACS C tube with 2 mL of TST buffer per sample. Keep tubes on wet ice.'

Transfer a ~1x1x1 mm piece of tissue directly into the buffer, making sure that the tissue is not stuck to any walls in the tube. Return tubes to ice immediately after transferring tissue.

If the tissue piece is significantly larger than the recommended size, a smaller piece should be cut on dry ice for nuclei isolation. Multiple pieces can be cut for tissue that looks heterogenous.

Once all tissue pieces are transferred into buffer, invert the tubes with a firm flick.

Tissue should be floating freely in buffer and not caught on any walls of the tube or tube cap.

Secure the C tubes to the GentleMACS Dissociator and run the m_spleen_01.01 program. After it completes, repeat the program a second time for a total of two runs on each tissue sample.
3. First Centrifugation

Detach the tubes from the GentleMACS Dissociator.
Incubate the samples on wet ice for 5 minutes (upside down).

Centrifuge the tubes for 2 minutes at 500g at 4ºC to collect the suspension and remove foam. Set the acceleration to 10 and deceleration to 5.
Immediately following the centrifugation, resuspend the nuclei pellet in the supernatant within the same C tube.
4. First Filtration

Prepare a 15 mL Falcon tube on wet ice with a a 40 um Falcon Cell Strainer. Wash the filter with 1 mL of 1x ST buffer with RNase inhibitors.

Transfer the homogenized ~2 mL suspension of nuclei into the filter.

Wash the used C tube with 1 mL of 1x ST buffer with RNase inhibitors, then transfer into the filter. Wash the filter with an additional 1 mL of 1x ST with RNase inhibitors.
The total volume of suspension should be about 5 mL.
5. Second Centrifugation

Centrifuge in a swinging bucket rotor for 10 minutes at 500 g at 4ºC.
The longer centrifugation helps recover more nuclei in a pellet than 5 minutes.

If there is no pellet observed, transfer to a smaller tube and centrifuge again for 10 minutes. Then follow the steps below.

Carefully transfer the supernatant to a new, labeled 15 mL Falcon and set aside on wet ice.
If yield is low, more nuclei may be recovered by spinning down the supernatant again.

Resuspend the remaining nuclei pellet in 50-70 μL of PBS + BSA (1%) + RNase Inhibitors solution.
Volume can be adjusted based on pellet size and desired concentration.
6. Second Filtration

Prepare a 5 mL FACS tube with filter cup on wet ice.

Carefully transfer the nuclei suspension through the new filter.
To pass a small volume through the filter, it can be helpful to hold the pipette tip against the membrane of the filter and apply gentle pressure while dispensing.
If necessary, the suspension can be carefully pulled through to the other side of the filter using a clean pipette tip.
7. Quality Control

Count nuclei using INCYTO C-Chip Neubauer Improved Disposable Hemocytometers and dilute to desired concentration if necessary.

  
  
8. Loading

Load sample on 10X (recommended to load between 8,000 -10,000 nuclei per 10X channel). For multiplexing of 4 samples, load 32,000- 40,000 per 10X channel.