Human Fallopian Tube and Ovary Dissociation for Single Cell RNA-Seq
Melissa Javellana, Mark Eckert, Ernst Lengyel
Abstract
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
Solution Preparation
Prepare 10mL of Pronase solution per sample in 50ml LoBind conical :
| A | B |
|---|---|
| Reagent | Quantity |
| Opti-MEM | 10 ml |
| Pronase (~7 U/mg) | 18 mg |
Prepare 10mL of digestion buffer per sample in a 50 mL LoBind conical:
| A | B |
|---|---|
| Reagent | Quantity |
| HBSS | 10 ml |
| Collagenase IV (>120 U/mg) | 15 mg |
| Hyaluronidase (750-3000 U/mg) | 10 mg |
| DNaseI (20,000 U/ml) | 10 μl |
Note: Keep all solutions at 37°C throughout protocol.
Dissociation
Rinse gross blood off samples with DMEM/10% FBS.
Fallopian tube isthmus: bivalve
Fallopian tube ampulla: bivalve
Fallopian tube fimbriae : leave whole
Ovary: mince 100mg tissue into 1-2 mm pieces.
Place each sample into 10mL of Pronase solution . Place in 37°C orbital shaker at 200 rpm for 0h 30m 0s.
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 10mL 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.
Place remaining solid fraction in digestion buffer . Place on 37°C orbital shaker at 200 rpm for 0h 30m 0s to 0h 45m 0s
Spin down epithelial fraction at 400 rcf for 0h 4m 0s. Aspirate and discard supernatant. Re-suspend in 3mLDMEM/FBS and wait for solid tissue fraction to complete digestion.
Pass solid fraction solution through a 70 μm filter into conical containing epithelial fraction and rinse filter with 10mLDMEM/FBS.
Spin down at 400 rcf for 0h 4m 0s. Aspirate and discard supernatant. Re-suspend in 1mL PBS/0.05% BSA
RBC Clean-up
Move solution to Eppendorf tube and add 12µL 0.5Molarity (M) EDTA
Vortex the EasySep RBC depletion reagent bottle for 0h 0m 30s to re-suspend
Add 25µL reagent and gentle vortex for 0h 0m 3s until solution appears homogeneous
Place on magnet and incubate 0h 5m 0s
Leaving on magnet, pipet off clear solution
Re-suspend in pre-warmed 37°C DMEM/10%FBS.
Assess viabilty with trypan blue if proceeding immediately to single cell library creation or method of choice if optimizing protocol.
