TITLE: Single Cell Isolation of Human Lung Organoids
AUTHORS: Morris Baumgardt, Maren Hülsemann, Anna Löwa, Benedikt Obermayer, Emanuel Wyler, Stefan Hippenstiel, Andreas C. Hocke, Katja Hönzke

[DESCRIPTION]
This protocol describes the single cell isolation of infected human alveolar-like organoids in order to perform single cell RNA sequencing or flow cytometry. The protocol focussed on the detailed preparation of samples to prepare single cells for further processing according to the protocols from 10x Genomics for single cell sequencing. The described steps also include the optional fixation of single cells with methanol. In that case, the protocol describes how to rehydrate single cell after methanol fixation to process them for single cell sequencing. 

[GUIDELINES]
This protocol describes the processing of human alveolar-like organoids which have been grown according to Youk et al., 2020. https://doi.org/10.1016/j.stem.2020.10.004

[STEPS]
SECTION: Single cell dissociation
1. Remove organoid medium from two wells and add 1 mL cold base medium per well (300,000 cells per well is a good amount to have sufficient cells even after the dissociation process.

SECTION: Single cell dissociation
2. Transfer organoids with a 1000 µL pipette to a 2 mL reagent tube and spin down at 300 x g, 5 min, 4 °C.

SECTION: Single cell dissociation
3. Very carefully discard the supernatant with a 1000 µL pipette.
SECTION: Single cell dissociation
4. Resuspend the cell pellet in 1 mL TrypLE Express/well (initally combined).

SECTION: Single cell dissociation
5. Incubate at 37 °C for 15 min carefully vortex every 5 mins, stop the reaction with 1 mL of base medium.

SECTION: Single cell dissociation
6. Spin down at 300 x g, 5 min, 4 °C.

SECTION: Single cell dissociation
7. Very carefully discard the supernatant.
SECTION: Single cell dissociation
8. Resuspend the cell pellet in 1 mL cold base medium.

SECTION: Single cell dissociation
9. Resuspend the whole supernatant 3 times with 27G needle and 1 mL syringe.
SECTION: Single cell dissociation
10. Filter cells through the cap of FACS tubes (40 µm pore size).
SECTION: Single cell dissociation
11. Count cells with the use of Trypan Blue staining. Make sure a single cell solution is provided. According to 10x Genomics recommendation a high-quality cell suspension has high cell viability (>90% ideally, >70% is acceptable), low cell debris and low cell clumping.

  

SECTION: Single cell dissociation
12. Proceed with methanol fixation or prepare samples immediately according to 10x Genomics protocol for RNA-Sequencing or flow cytometry.


 


   
The Cell Ranger Software Suite (Version 3.1.0) was used to process raw sequencing data with the GRCh38 reference for the control samples, and GRCh38 augmented by the SARS-CoV-2 (NC_045512.2). Viral transcripts including 3'UTR sequences were extracted from the Genbank records and added to the Gencode v33 reference. We used CellBender to remove background RNA and scrublet to identify doublets. Single-cell RNA sequencing data analysis was performed in R (version 3.6.3) with Seurat (version 3.2.1). Cells with at least 500 and less than 5000 detected genes and less than 10% mitochondrial content were combined from each library and library depth (total number of UMIs) was regressed out when scaling data. We additionally regressed out cell cycle scores calculated with Seurat's CellCycleScoring function. After automated clustering, DoubletFinder was used to again identify likely cell doublets, and cluster annotation was performed with Seurat's ‘TransferData’ workflow using the Human Lung Cell Atlas reference dataset, using only epithelial cells for the organoid data.
SECTION: Methanol fixation (optional)
13. In case of scRNA-sequencing: Ideally methanol fixation should be avoided and the samples should be immediately processed for library preparation according to the 10x Genomics protocol  (Chromium Next GEM Single Cell 3’ Reagent Kits v3.1 (Dual Index) with Feature Barcode technology for Cell Multiplexing User Guide).
SECTION: Methanol fixation (optional)
14. Centrifuge the dissociated cells at 300 x g, 5 min, 4 °C.

SECTION: Methanol fixation (optional)
15. Remove the supernatant without disrupting the cell pellet.
SECTION: Methanol fixation (optional)
16. Add 1 mL 1x PBS and gently resuspend pellet 10 times with 1000 µL pipette tip or until cells are resuspended. Each tube should have 1-2 x106 cells (1 well of organoids usually contains up to 0.5 x106 cells).

SECTION: Methanol fixation (optional)
17. Repeat washing steps 14, 15 and 16.
SECTION: Methanol fixation (optional)
18. 
Centrifuge at 300 x g, 5 min, 4 °C.
SECTION: Methanol fixation (optional)
19. Remove the supernatant without disrupting the cell pellet.
SECTION: Methanol fixation (optional)
20. Add 200 µL chilled 1x PBS/1x106 cells and gently resuspend pellet 10x with 1000 µL pipette tip or until cells are resuspended.

SECTION: Methanol fixation (optional)
21. Add 800 µL chilled 100% methanol/1x106 cells. To avoid clumping of cells, add methanol drop by drop while gently stirring the cell suspension with the pipette tip in the tubes.

 

SECTION: Methanol fixation (optional)
22. Incubate for 30 min at -20 °C.
SECTION: Methanol fixation (optional)
23. Export from the BSL3 and store fixed cells either at -20 °C or -80 °C (stability has been shown for both termperatures for up to 6 weeks).

 

SECTION: Rehydration of methanol-fixed cells
24. Place the microcentrifuge tube containing the methanol-fixed cells on ice to equilibrate to 4 °C for 5 min.

SECTION: Rehydration of methanol-fixed cells
25. Centrifuge fixed cells at 1000 x g, 5 min, 4 °C.

SECTION: Rehydration of methanol-fixed cells
26. Remove the supernatant without disrupting the cell pellet.
SECTION: Rehydration of methanol-fixed cells
27. Based on starting cell concentration and assuming ~50% cell loss, add an appropriate volume of Wash-Resuspension Buffer to obtain a concentration of 700-1,200 cells/µL. Gently pipette mix using a regular-bore pipette tip until a single cell suspension is achieved.
SECTION: Rehydration of methanol-fixed cells
28. Pass the sample through a 40 μm Flowmi Cell.
SECTION: Rehydration of methanol-fixed cells
29. Count cells.
SECTION: Rehydration of methanol-fixed cells
30. Proceed immediately with the 10x Genomics Single Cell protocols. Delay in proceeding may result in RNA loss.