Preparation of single cell suspensions from human intestinal biopsies for single cell genomics applications
Ran RZ Zhou, Oni Basu
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Abstract
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.
Steps
Pre-Dissociation
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
Sample Wash before dissociation
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.
Repeat step 2 three more times or until the PBS becomes clear.
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.
0h 1m 0s On ice
Dissociate Epithelial layers from Lamina propria
Add 3 ml ice-cold Dissociation media to each 5 ml tube.
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 0h 15m 0s
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.
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.
Epithelium Digestion
Centrifuge the epithelium in the supernatant in 5 ml conical tubes at 300 g x 5 mins at 4 celsius.
300x g,4°C
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.
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.
Centrifuge at 300 g x 5 mins at 4 celsius.Remove supernatant. Resuspend cells in 1 ml wash media and place on ice.
300x g,4°C
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.
Lamina propria Digestion
Wash the tissue one times with 3 ml Wash media. Aspire the Wash media as much as possible.
Add 2 ml Digest media. Incubate the tissue at 37°C with end-over-end rotation 20 rpm for up to 30 mins.
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.
Centrifuge at 300 g x 5 minus at 4 Celsius. Remove supernatant and suspend cells in 1 ml wash media.
300x g,4°C
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.
Red blood cell removal
Dilute one volume of cell suspension (epithelium or lamina propria; keep tissue types separate) by two volumes of Red Blood Cell Lysis Solution.
Gently mix by inverting the tubes and incubate for 2 minutes at room temperature.
Centrifuge at 300 g for 5 minutes at 4 Celsius.
300x g,4°C
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.
Cell Suspension Preparation
Count the cells with Trypan Blue staining.
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).
