TITLE: Nuclei isolation from frozen gonad tissue
AUTHORS: Maria del Carmen Sancho Serra or Carmen Sancho Serra

[DESCRIPTION]
This protocol describes  how to perform nuclei isolation from frozen gonad tissues using Dounce homogenizers. It can be used as well for ovary strips frozen in OCT

[GUIDELINES]
Human tissue and cells from primary samples may contain uncharacterized adventitious agents, including blood-borne viruses. No attempt will be made to culture these agents deliberately. Correct use of PPE will drastically reduce the risks. 
Tissue collection for this protocol requires prior approval by the users' Institutional Ethics Board or equivalent ethics committee

[STEPS]
SECTION: Buffer preparation
1. Nuclei Isolation Buffer 1 (NIM1)

Can be made, filtered and stored at 4°C for up to 6 months

 
Nuclei Isolation Buffer 2 (NIM2)

To be made and used on the day of experiment, keep on ice.
Pre-dilute 1M DTT: 5 ml 1 M DTT in 4.995 ml water.
For 100x Protease inhibitor stock, dissolve 1 tablet in 500 ml nuclease free water. Stock solution can be stored at 2 to 8 °C for 1 to 2 weeks, or at least 12 weeks at -15 to -25 °C.


 

Homogenization Buffer

To be made and used on the day of experiment, keep on ice. Do NOT vortex.

 

Wash Buffer


 

Diluted Nuclei Buffer 

Prepare this buffer while centrifuging nuclei in step 11


 

90% Percoll solution

Prepare fresh and keep on ice

 

2. Clean Dounce homogenizers and pestle B with 70 % ethanol, then Milli-Q water. Place on ice with 50 ml Falcon tube to pre-chill
3. Collect tissue on dry ice from -80 °C freezer and proceed immediately 
4. Transfer tissue biopsy to homogenizer using sterile, disposable tweezers. If the piece of tissue is in OCT, cutted slides should be directly transferred to the dounce homogenizer already seating on dry ice
5. Mix Homogenization buffer and transfer 3 mL to the homogenizer making sure to wash the tissue to the bottom of the tube. Wait until buffer is liquid again

16. Follow 10X Single Cell Multiome ATAC + Gene Expression kit protocol (isothermal incubation + GEM generation and barcoding + GEM incubation + quenching reaction)
Keep GEMs at -80 °C 

6. Gently homogenize the tissue using the tight pestle (B). Up and down but do not grind. Try to avoid bubbles. 10-20 strokes depending on how the tissue behaves. Rinse the pestle with 0.5 mL of Homogenization buffer. Place the pestle in Virkon for cleaning.

7. Pour homogenate through a 40 µm cell strainer into 50 ml Falcon tube on ice. Rinse homogenizer with additional 0.5 mL  Homogenization buffer and pour it through the strainer. Make sure to transfer droplets of the filtrate from the under side of the strainer using a pipette.

Glass homogenizers should be left for 24 hours in Virkon, then cleaned with 70 % ethanol and Milli-Q water.
8. Centrifuge the 50 ml Falcon tube @ 500 g, 6 mins, 4 °C with acceleration set at 0 and deacceleration set to 3 in Eppendorf centrifuge.
9. Carefully remove most of the supernatant with 1 ml pipette tip. Leave a small amount and gently resuspend the nuclei using a wide bore tip (optional). Add 0.5 mL of Wash buffer and carefully resuspend nuclei and transfer to an 1.5 ml eppendorf (NOT low bind).  
11. Centrifuge at 500 g, 3 mins, 4 °C. During centrifuge time, prepare Diluted Nuclei Buffer

Remove all supernatant and resuspend nuclei in the remaining (~100 µl) wash buffer. Check  how is the nuclei quality using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension)


13. Check the nuclei in the new buffer using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension). Normally will appear clumpy in this buffer!!!! 
 
12. 
Skip filtering with Flowmi filter if there is not a lot of debris. 

Adjust here depending on the size and appearance of the nuclei. If there is enough nuclei density, resuspend in  100 µL  of Diluted Nuclei Buffer

14. Filter with a 40 µm Flowmi cell strainer and check nuclei suspension again (a little bit less volume due to filtering)
15. Count nuclei using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension). Concentrate down if necessary, centrifuging at 500 g, 3 mins, 4 °C and taking out extra supernatant volume according to desirable nuclei stock concentration versus targeted nuclei recovery.
10. Check  how is the nuclei quality using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension)
10.1. If the nuclei solution is clean enough and not many debris present, add 0.5 mL more of Wash buffer and go to step 12

10.2. If many debris is present, start Percoll clean-up:

- Prepare 90% Percoll solution
- Prepare 2 epp with 475 µL of Wash Buffer
- Add 225 µL of the nuclei solution to each one of the epp
- Add 300 µL of 90% Percoll solution to each one of the epp
- Mix by inverting eppendorfs 5 times
- Centrifuge @ 20,000 g, 15 min, 4 °C
- Take supernatant without touching pellet that would be "floating" in the middle-lower part of the tube
- Add 500-900 µl Wash Buffer
- Centrifuge @ 500 g, 3 min, 4 °C
- Take as much supernatant as possible and add 500 µl Wash Buffer in each epp and combine them in one
- Centrifuge @ 500 g, 3 min, 4 °C
-Take most of the supernatant leaving 50-100 µl for resuspension.
- Count nuclei using a C-Chip (5 µl filtered Trypan blue, 5 µl nuclei suspension)
- Concentrate down if necessary, centrifuging at 500 g, 3 mins, 4 °C and taking out extra supernatant volume according to desirable nuclei stock concentration versus targeted nuclei recovery.
- Exemple if the nuclei stock is in100 µl: 
        add 5 µl 20x Nuclei buffer
        add 1 µl DTT(diluted 1:10 in water)
        add 2.5 µl RNase Inhibitor????