TITLE: Preparation of single cell suspensions from human intestinal biopsies for single cell genomics applications
AUTHORS: Ran RZ Zhou, Oni Basu

[DESCRIPTION]
The protocol is adapted from Fujii's and Smillies's reports for single cell transcriptome analysis from human intestines. It provides details on acquirement of single cell suspension from epithelium and lamina propria. This methods is modified to generate appropriate meterials from patient's intestinal biopsies for sinlge-cell transcriptome and genomic applications.

[BEFORE_START]
Generally two samples are obtained from each patient. The sample information from tissue containers (1.7 ml Eppendorf tube in our facility) are recorded. Each sample contains 2-3 pinches. Samples from the same patients but from different biopsy location (ileum and ascending colon) are processed separately. Wet weight of the tissues from each sample are measured by a micro-scale and recorded.

[GUIDELINES]
The human intestinal tissue are obtained after patients' consents and approval from Institutional Review Board at the University of Chicago (IRB Number: 15573A). All the samples are processed for research use only.

[STEPS]
SECTION: Pre-Dissociation
1. Chill 1x PBS, wash media and dissociation media on ice. Samples are transfered with Advanced DMEM/F12 based media in 1.7 ml eppendorf tubes on ice. Once received in lab, samples are transfered to 35 mm dish using sharp-end forceps. Alternatively, tissues can be transferred to a 5 ml conical tube using a P200 pipette.  
on ice 
 

SECTION: Sample Wash before dissociation
2. Place 3 ml ice cold PBS in each dish with each sample (containing 2-3 pinches). Shake the dish on ice to rinse off mucus. Alternatively, add 4 ml ice cold PBS to the 5 ml conical tube and invert the tube 5 times with the lid closed.


SECTION: Sample Wash before dissociation
3. Repeat step 2 three more times or until the PBS becomes clear.
SECTION: Sample Wash before dissociation
4. Transfer tissues from  the 35 mm dish to a clean 5 ml conical tube (tissues are remained in the same tube if washed in the conical tube). Add 0.2 ml Dissociation media and mince the tissues with Iris scissors for 1 minute on ice. 
 

1 min on ice 

SECTION: Dissociate Epithelial layers from Lamina propria
5. Add 3 ml ice-cold Dissociation media to each 5 ml tube. 
SECTION: Dissociate Epithelial layers from Lamina propria
6. place the tubes on ice on a linear rocker and rock the tubes (end to end) at 100/min for 60 mins. Alternatively rotate the tubes in a 37 Celsius oven for 15 mins. 
37 °C 15 min 

SECTION: Dissociate Epithelial layers from Lamina propria
7. Pipet the tissue in dissociation media in a 5 ml serological pipet 5 times and let the tissue settle in the tube on ice for 5 mins. Collect the supernatant and continue with Epithelium Digestion. Add 10 ml dissociation media to the tissue and repeat step 6. 
SECTION: Dissociate Epithelial layers from Lamina propria
8. Collected supernantant will be processed in the following section. Sedimented tissue represents the lamina propria and will be processed in the "Lamina propria Digestion" section.

 

SECTION: Epithelium Digestion
9. Centrifuge the epithelium in the supernatant in 5 ml conical tubes at 300 g x 5 mins at 4 celsius.
300 x g, 5 min, 4 °C 

SECTION: Epithelium Digestion
10. Remove supernatant and add 2 ml warmed TrypLE express to the pellet. Incubate the mixture at 37 celsius for 5 mins with rotation at 20 rpm.


 


SECTION: Epithelium Digestion
11. Neutralize TrypLE express by adding 2 ml wash media, agitate the tissue by pipetting up and down and filter through a 40 micron cell strainer. 

SECTION: Epithelium Digestion
12. Centrifuge at 300 g x 5 mins at 4 celsius.Remove supernatant. Resuspend cells in 1 ml wash media and place on ice. 

300 x g, 5 min, 4 °C  


SECTION: Epithelium Digestion
13. Take 20 ul cell suspension with 20 ul Tryplan blue and mix well. Check the cell viability on a light microscope. 
If viability is higher than 85%,  proceed with red blood cell removal.
 

SECTION: Lamina propria Digestion
14. Wash the tissue one times with 3 ml Wash media. Aspire the Wash media as much as possible.
SECTION: Lamina propria Digestion
15. Add 2 ml Digest media. Incubate the tissue at 37°C with end-over-end rotation 20 rpm for up to 30 mins.

 

SECTION: Lamina propria Digestion
16. Neutralize Liberase TM by 1 ml Quench media. Leave tissue on ice for 5 mins. Agitate the tissue by pipetting up and down and filter through a 40 micron cell strainer. 
SECTION: Lamina propria Digestion
17. Centrifuge at 300 g x 5 minus at 4 Celsius. Remove supernatant and suspend cells in 1 ml wash media. 
300 x g, 5 min, 4 °C 

SECTION: Lamina propria Digestion
18. Take 20 ul cell suspension with 20 ul Trypan Blue. Mix well and check the viability on a light microscope. Proceed with red blood cell removal.
SECTION: Red blood cell removal
19. Dilute one volume of cell suspension (epithelium or lamina propria; keep tissue types separate) by two volumes of Red Blood Cell Lysis Solution. 
SECTION: Red blood cell removal
20. Gently mix by inverting the tubes and incubate for 2 minutes at room temperature. 
SECTION: Red blood cell removal
21. Centrifuge at 300 g for 5 minutes at 4 Celsius. 
300 x g, 5 min, 4 °C 

SECTION: Red blood cell removal
22. Aspirate supernatant completely. Resuspend the cell pellet in 0.1 ml cell suspension buffer with RNase Inhibitor 0.1 U/ul (RNase Inhibitor is not needed if loading on 10x Genomics platform).  Proceed with Cell suspension preparation.
SECTION: Cell Suspension Preparation
23. Count the cells with Trypan Blue staining.

SECTION: Cell Suspension Preparation
24. Adjust cell density by cell suspension buffer with RNase Inhibitor 0.1 U/ul  (RNase Inhibitor is not needed if loading on 10x Genomics platform).