TITLE: Human Fallopian Tube and Ovary Dissociation for Single Cell RNA-Seq
AUTHORS: Melissa  Javellana, Mark  Eckert, Ernst Lengyel

[DESCRIPTION]
This protocol provides a procedure for human fallopian tube and ovary dissociation into single cell suspension prior to single cell RNA-sequencing. It involves removing the epithelial fraction and then further digesting the remaining solid tissue fraction to gain maximum yield from all tissue compartments.


[BEFORE_START]
Prepare DMEM/10%FBS stock
Prepare 0.5M EDTA stock
Prepare 1mL PBS/0.05% BSA per sample


Note: Our samples are retrieved from the OR immediately upon removal from patient and transported to pathology. The pathologist grossly examines the sample and provides 100-200mg transverse tissue segments from the isthmus, ampulla and fimbriae of the fallopian tube and a longitudinal segment from the center of the ovary. Tissue segments are transported to the lab in DMEM/10% FBS.

[STEPS]
SECTION: Solution Preparation
1. Prepare 10 mL of Pronase solution per sample in 50ml LoBind conical :



 

 

SECTION: Solution Preparation
2. Prepare 10 mL of digestion buffer per sample in a 50 mL LoBind conical:

 

SECTION: Dissociation
3. Rinse gross blood off samples with DMEM/10% FBS. 


SECTION: Dissociation
4. Place each sample into 10 mL of Pronase solution. Place in 37 °C orbital shaker at 200 rpm for 30 min.

SECTION: Dissociation
5. Using a 10 ml pipette, triturate tissue to remove loose epithelial fraction. Pass all of the pronase solution  through 70 μm filter into new 50 ml LoBind conical and rinse filter with 10 mL DMEM/10%FBS. Recover solid fraction from original conical and/or  off of filter.

NOTE: You now have an epithelial fraction and a solid fraction. You will continue to process the epithelial fraction and solid fraction separately in the next steps. Both contain potentially unique cell types.
SECTION: Dissociation
6. Place remaining solid fraction in digestion buffer. Place on 37 °C orbital shaker at 200 rpm for 30 min to 45 min 

SECTION: Dissociation
7. Spin down epithelial fraction at 400 rcf for 4 min. Aspirate and discard supernatant. Re-suspend in 3 mLDMEM/FBS and wait for solid tissue fraction to complete digestion.

SECTION: Dissociation
8. Pass solid fraction solution through a 70 μm filter into conical containing epithelial fraction and rinse filter with 10 mLDMEM/FBS.

SECTION: Dissociation
9. Spin down at 400 rcf for 4 min. Aspirate and discard supernatant. Re-suspend in 1 mL PBS/0.05% BSA 

SECTION: RBC Clean-up
10. Move solution to Eppendorf tube and add 12 µL 0.5 Molarity (M) EDTA

SECTION: RBC Clean-up
11. Vortex the EasySep RBC depletion reagent bottle for 30 s to re-suspend 

SECTION: RBC Clean-up
12.  Add 25 µL reagent and gentle vortex for 3 s until solution appears homogeneous

SECTION: RBC Clean-up
13. Place on magnet and incubate 5 min 

SECTION: RBC Clean-up
14. Leaving on magnet, pipet off clear solution
SECTION: RBC Clean-up
15. Re-suspend in pre-warmed 37 °C DMEM/10%FBS.

SECTION: Solution Preparation
2.1. Note: Keep all solutions at 37 °C throughout protocol.

SECTION: Dissociation
3.1. Fallopian tube isthmus: bivalve
SECTION: Dissociation
3.2. Fallopian tube ampulla: bivalve
SECTION: Dissociation
3.3. Fallopian tube fimbriae: leave whole 
SECTION: Dissociation
3.4. Ovary: mince 100 mg tissue into 1-2 mm pieces.
SECTION: RBC Clean-up
16. Assess viabilty with trypan blue if proceeding immediately to single cell library creation or method of choice if optimizing protocol.