TITLE: BIDMC-TMC Nuclei Isolation from Frozen Cervix for Single Cell RNA-Seq
AUTHORS: Luciano G Martelotto, Antonella Arruda de Amaral, Nikolaos Kalavros, Ioannis Vlachos, Shuoshuo Wang

[DESCRIPTION]
As a fibromuscular organ, human cervix samples are naturally resilient to withstand strains and deformation during pregnancy and birth. Due to its consistency and composition, dissociation and disrupting the cervix or other parts of uterus to extract nuclei is a challenging step for single cell applications.

Here, we present a comparably simple and fast protocol for single nucleus RNA sequencing that has been developed at BIDMC TMC, Spatial Technologies Unit of the Beth Israel Deaconess Medical Center of HuBMAP Consortium in collaboration with Luciano Martelotto from the University of Adelaide. 

We optimized this protocol for human cervix and other uterine parts, but also tested it successfully for other tissue types very rich (90%) in fibrous connective tissues. Our protocol combines liquid nitrogen facilitated tissue disruption with the fixed RNA (flex) kit from 10x Genomics and generating high quality libraries for HuBMAP.

[BEFORE_START]

Get liquid nitrogen. 1 liter suffices for preparation of 2-4 sample. Industrial dry ice is very dirty and should not be used as substitute.
Pre-cool centrifuge and other utensils.
Take precautions when using alternatives, such as thermal blocks instead of ThermoMixer with heated lid, wrong filters. 

[STEPS]
SECTION: Sample Preparation
3. Ensure the samples are free of OCT if they were previously embedded. Coarsely trim off the peripheral OCT and rinse the sample core with RNase-free chilled PBS thoroughly before processing.

SECTION: Sample Preparation
4. Pre-cool a mortar and pestle by filling with sufficient liquid nitrogen;
SECTION: Sample Preparation
5. Place the tissue on a pre-chilled 6cm or 10cm dish maintained on a cold plate, and mince tissue finely with a sterile razor blade to facilitate the fixative penetration. Do not over-mince. Transfer the minced tissue into the mortar; 
SECTION: Sample Preparation
6. Add enough liquid nitrogen to submerge the whole sample and wait until the tissue is fully frozen, then grind the tissue carefully to generate a granular fine power.
SECTION: Sample Preparation
7. Using a sterile disposable spatula, transfer the suspension of tissue powder carefully into a pre-cooled 1.5 or 2ml low-bind and safe-lock Eppendorf tube and allow liquid nitrogen to evaporate without thawing the sample powder. The exact process needs to be optimized depending on the tissue composition. 

For immediate processing, transfer the powder to the pre-filled 1.5 mL tube containing the Fixation Buffer. Pipette mix with a wide-bore pipette tip.

Alternatively, the powder can be stored at -80°C in an Eppendorf Tube or a cryovial for extended period.

 

SECTION: Sample Preparation
6.1. Repeat this step until the tissue becomes a fine powder. Ensure the tissue never thaws throughout this process.
SECTION: Sample Fixation
8. Incubate atRoom temperature for at least 60 min, or at 4 °C    .

SECTION: Sample Fixation
8.1. Gently invert the sample tube to ensure contact between Fixation Buffer and powered samples.
SECTION: Sample Dissociation
10. Prepare the Sample Quenching Buffer as described above;
SECTION: Sample Dissociation
9. Centrifuge the samples at  850 rcf, 5 min .

SECTION: Sample Dissociation
11. Cool the centrifuge to 4°C; 
SECTION: Sample Dissociation
12. Remove the supernatant and re-suspend the pellet in Quenching Buffer; 
From this moment forward the sample should be kept on ice;
SECTION: Sample Dissociation
13. Centrifuge the samples again at 850 rpm, 5 min, 4 °C .

SECTION: Sample Dissociation
14. Remove the supernatant, and add 200 µL (or enough to cover the tissue) of   .

SECTION: Sample Dissociation
15. Homogenize the tissue using a small plastic pellet pestle to ensure complete dissociation;
SECTION: Sample Dissociation
16. Add more EZ Lysis buffer for a total of 1 mL , incubate for 10 min on ice;
 


SECTION: Sample Cleanup
17. Prepare the Sample Wash Buffer and the Sample Resuspension Buffer as described above;
SECTION: Sample Cleanup
18. Centrifugate the samples at 850 rpm, 5 min, 4 °C 

SECTION: Sample Cleanup
19. Remove the supernatant and re-suspend in 800 µL  of Wash Buffer;

SECTION: Sample Cleanup
20. Filter the resuspension through a 70µm sterile filter and into a 2 mL Eppendorf LoBind tube. Do not discard original tube.

SECTION: Sample Cleanup
21. Add 800 µL more of wash buffer to the original tube and wash the walls. Pass this through the same filter to increase nuclei capture;

SECTION: Sample Cleanup
22. Centrifugate the samples at 850 rpm, 5 min, 4 °C 
SECTION: Sample Cleanup
25. After the final centrifugation, resuspend pellet in 1 mL of Resuspension Buffer;

 

SECTION: Sample Cleanup
26. Filter final nuclei suspension through a 30µm sterile filter and into a 1.5 mL LoBind Eppendorf tube.
SECTION: Sample Cleanup
27. Count the nuclei, and proceed to immediately to 10X protocol CG000691 (see Ref.);
 

SECTION: Sample Cleanup
23. Remove the supernatant and re-suspend in 1 mL  of Wash Buffer;
SECTION: Sample Cleanup
24. Repeat steps 21 and 22 at least one more time for a total of 3 washes.
 

SECTION: Sample Storage
28. Thaw Enhancer (10x Genomics PN-2000482) for 10 minutes at 65°C;
SECTION: Sample Storage
29. Samples can be kept for up to 1 week at 4°C with 0.1 volume of Enhancer;
SECTION: Sample Storage
30. Samples can be kept for up to 6 months at -80°C with 0.1 volume of Enhancer, and 10% volume of 50% Glycerol.
SECTION: Buffer Preparation
1. 
 

SECTION: Buffer Preparation
2. Prepare fresh buffers based on CG000478, page 2, including Fixation Buffer, Quenching Buffer and Wash-Resuspension buffer. 1 ml Fixation Buffer will be used per sample. Prepare the Sample Fixation Buffer as described above and aliquot 1mL into a 1.5mL Eppendorf Tube;

Fixation Buffer – 1mL per Sample – Room Temp.
 
Quenching Buffer – 1 mL per Sample – 4°C
 
Tissue Wash Buffer – 4mL per Sample – 4°C
 
Tissue Resuspension Buffer  – 1 mL per Sample – 4°C