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

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Pre-Dissociation]
Chill 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 after the media are chilled. Alternatively, tissues can be transferred using a P200 pipette.
on ice
Pinched tissues range from 6 to 20 mm3 in size.
?. [Sample Wash before dissociation]
Place 3 ml ice cold wash media in each dish with each sample (containing 2-3 pinches). Shake the dish on ice to rinse off mucus.
?. [Sample Wash before dissociation]
Repeat step 2 three more times or until the wash media becomes clear.
?. [Sample Wash before dissociation]
Transfer tissues from  the 35 mm dish to a clean 5 ml conical tube. Add 0.2 ml Dissociation media and mince the tissues with Iris scissors for 1 minute on ice.
The minced tissues range from 1 to 3 mm3 in size, can easily pass a P1000 tip.
on ice
?. [Dissociate Epithelial layers from Lamina propria]
Add 3 ml ice-cold Dissociation media to each 5 ml tube.
?. [Dissociate Epithelial layers from Lamina propria]
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.
on ice
4 °C
?. [Dissociate Epithelial layers from Lamina propria]
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.
?. [Dissociate Epithelial layers from Lamina propria]
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.
The epithelial sheets isolated from lamina propria remain in the Dissociation media supernatant. The lamina propria will be processed separately from epithelium.
?. [Epithelium Digestion]
Centrifuge the epithelium in the supernatant in 5 ml conical tubes at 300 g x 5 mins at 4 celsius.
Centrifuge: 300 34, 5 min, 4 10
?. [Epithelium Digestion]
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.
A hybridation oven with a rotisserie is recommended.
?. [Epithelium Digestion]
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.
?. [Epithelium Digestion]
Centrifuge at 300 g x 5 mins at 4 celsius.Remove supernatant. Resuspend cells in 1 ml wash media and place on ice.
Centrifuge: 003 34, 5 min, 4 10
?. [Epithelium Digestion]
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.
Either hemocytometer or Countess automated cell counter can be used for viability assessment. Live cells are excluded from staining blue and the numbers of live and dead cells are both counted for viablity rate = 100% x N of live cells/(N of live cells + N dead cells). At least 50 cells are counted in total.
?. [Lamina propria Digestion]
Wash the tissue one times with 5 ml Wash media. Aspire the Wash media as much as possible.
?. [Lamina propria Digestion]
Add 3 ml Digest media. Incubate the tissue at 37°C with end-over-end rotation 20 rpm for up to 30 mins.
Check the digestion after 20 mins incubation with Digest media. If cell clumps remains and cell viability  > 95%, incubate for additional 5 minutes.
?. [Lamina propria Digestion]
Neutralize TrypLE by 0.5 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.
?. [Lamina propria Digestion]
Centrifuge at 300 g x 5 minus at 4 Celsius. Remove supernatant and suspend cells in 1 ml wash media.
Centrifuge: 300 34, 5 min, 4 10
?. [Lamina propria Digestion]
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.
?. [Red blood cell removal]
Gently mix by inverting the tubes and incubate for 2 minutes at room temperature.
?. [Red blood cell removal]
Centrifuge at 300 g for 5 minutes at 4 Celsius.
Centrifuge: 300 34, 5 min, 4 10
?. [Red blood cell removal]
Aspirate supernatant completely. Resuspend the cell pellet in 0.1 ml cell suspension buffer with RNase Inhibitor 0.1 U/ul. Proceed with Cell suspension preparation.
?. [Cell Suspension Preparation]
Count the cells with Trypan Blue staining.
?. [Cell Suspension Preparation]
Adjust cell density by cell suspension buffer with RNase Inhibitor 0.1 U/ul.