May 21, 2026

Single cell dissociation of intestinal gut tissue for scRNAseq_v2

 Forked from Single cell dissociation of intestinal gut tissue for scRNAseq of all cell types
DOI
https://dx.doi.org/10.17504/protocols.io.e6nvwwwrdvmk/v1
  • Mennatallah Ghouraba1,
  • Rebecca McIntyre2
  • 1Wellcome Sanger institute;
  • 2Wellcome Sanger
  • MennaGH
Mennatallah Ghouraba
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DOI: https://dx.doi.org/10.17504/protocols.io.e6nvwwwrdvmk/v1
Protocol Citation: Mennatallah Ghouraba, Rebecca McIntyre 2026. Single cell dissociation of intestinal gut tissue for scRNAseq_v2. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwwwrdvmk/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 18, 2026
Last Modified: May 21, 2026
Protocol  Integer ID: 317286
Keywords: scrnaseq of intestinal biopsy, isolation of healthy intestinal epithelial cell, single cell dissociation of intestinal gut tissue, healthy intestinal epithelial cell, intestinal biopsies on ice, comparable to collagenase digest, collagenase digest, warm collagenase digest method, intestinal biopsy, warm collagenase digest, intestinal gut tissue, enteroendocrine cell, collagenase iv, bacillus licheniformis protease, epithelial cell, minimise gene expression change, single cell dissociation, scrnaseq, minimise changes to gene expression, gene expression, inhibitors of anoiki, mesenchymal cell, digest, start of the digest, changes in gene expression, pan caspase inhibitor, immune cell, major cell type, cell viability
Abstract
This protocol was developed for scRNAseq of intestinal biopsies. For our study, we needed to preserve all cell types and minimise changes to gene expression, while keeping all cells healthy in order to detect small genotype-associated changes in gene expression (expression quantitative trait loci).

To achieve this, we use the Bacillus Licheniformis Protease (BLP) which digests intestinal biopsies on ice. BLP does not release mesenchymal cells very well, and so Collagenase IV is used at RT for 10 min to release more of those. We add two inhibitors of anoikis - Rocki and a pan caspase inhibitor - at the start of the digest. No digests are performed at 37 oC to avoid anoikis and minimise gene expression changes. Finally, cells are incubated in a hypotonic solution to remove low quality cells that are pro-apoptotic.

Expected results: Trypan blue staining shows cell viability is comparable to Collagenase digests (90%). It takes another ~1 h for cells to pass through the Chromium. After 10X, we see a substantial increase in the viability of epithelial cells compared to warm collagenase digest methods. The digest supports the isolation of healthy intestinal epithelial cells, which are usually of low quality as they are prone to anoikis during standard EDTA incubation or warm collagenase digests. All major cell types are identified, including rare tuft cells and enteroendocrine cells. Although detectable, mesenchymal cells are not as abundant as they are after warm collagenase digests.
Guidelines
Follow local research ethics committee guidelines on the collection and use of human tissue for research purposes.
Safety warnings
All experiments should be carried out in a CL2 microsafety cabinet
Equipment and disposables

ItemCatalogue no.
Micro-scissors (straight vannas) VWR 233-1103
HaemocytometerC-chip NanoEnTek, DHC-N01
30 µm strainers (CellTrics 04-0042-2316)CellTrics 04-0042-2316
40 µm cell strainer Falcon 352340
Large centrifuge (for 15 ml tubes) set to 4oC
15 ml and 50 ml falcon tubes
6 well plate or 35 mm plate
Nuclease free microfuge tube

Sample collection buffer

ReagentCatalogue no.PreparationStorage
HBSS without Ca2+ and Mg2+ (-/-)e.g. Gibco, 14175053NA4°C

BLP digest buffer (make on ice)

ReagentCatalogue no.PreparationStorage
EDTAe.g. Lonza, 51201NART
HBSS (without Ca2+ and Mg2+; -/-)e.g. Gibco, 14175053NART
Bacillus Licheniformis Protease (BLP)Sigma, P5380-250 mg1300 U/ml (10 X) in HBSS-/- (usually ~2.5 ml). Make 130U aliquots (usually ~50 µl). 4°C
Y-27632 dihydrochloride, Rho kinase inhibitor (Rocki). MW 320Abcam, ab120129 -10 mg10 mM (200X) = 10 mg in 3 ml waterPowder 4°C Aliquots @ -20°C (for 6 months) Aliquots @4oC for up to 1 week. Equilibrate to RT 1 h before.
QVD-OPh (caspase-3, 1, 8 and 9 inhibitor). MW 513.5 Abcam, ab141421 - 1 mg1 mM (200X) = 1 mg in 1.95 ml DMSO Powder -20°C Aliquots @ -20 °C for up to 1 year. Aliquots @ 4oC for up to 1 week. Equilibrate to RT 1 h before making aliquots.
Digest buffer2mM EDTA: 200µl 0.5M EDTA + 49.5 ml HBSS-/-4°C
BLP digest bufferPer sample: 500 µl Digest buffer + 2.5 𝛍L Rock-I + 2.5 𝛍L QVD-0Ph + 50 𝛍L BLP 4°C.Make fresh, dispose if not used

Collagenase IV + DNAse I digest buffer (room temperature)

ReagentCatalogue no.PreparationStorage
EDTALonza,51201N/ART
HBSS (with Ca2+ and Mg2+; +/+)Gibco, 14065056N/A4°C
1M CaCl2Sigma 21115-100 mlN/ART
Collagenase IVWorthington LS004188 - 100 mg50 U/µl in HBSS+/+. Make 20 µl aliquotsPowder -20 °C. Aliquots @-80. Thaw on ice.
DNAse ISTEMCELL Tech 079001 mg/mlAliquots -20 °C. Thaw on ice.
5mM CaCl2 buffer Add 250 µL 1M CaCl2 to 49.5ml HBSS+/+. RT
Collagenase IV, DNAse I digest bufferMake buffer master Per sample: 435 µL CaCl2 buffer + 50 µL DNase I (0.1mg/mL)+ 15 µL Collagenase IV (750U) RT

Wash buffer (make on ice)

ReagentCatalogue no.PreparationStorage
Bovine Serum Albumin (BSA)Sigma A9543 – 5gMake 25% BSA by adding 5 g and top up to 20 mL with HBSS-/-Powder 4°C. Aliquots -20°C Thaw on ice before use.
Wash buffer1% (v/v) BSA in HBSS-/- i.e. add 48 ml to 2 ml 25% BSA4°C (Discard after 7 days)

Other reagents

ReagentCatalogue no.PreparationStorage
ACK lysis buffer Gibco A10492-01NART
Trypan blueNART

Method
Method is for two TI biopsies (3 mm) or three rectum biopsies, which are tougher to dissociate. If you are unable to process the biopsies immediately, we have had good success with cryopreserving in 90% HI FBS and 10% DMSO. -Samples are collected into 500 µL collection buffer and kept on ice.
-Samples generally begin processing, either to digest or cryopreserve, 30-60 min after collection.
-Set centrifuge to 4°C.
-All steps are performed on ice unless otherwise stated.
-After any wash step, it is best to pour off the supernatant and allow the last drop to drip out (flick it!), before re-suspending in the residual buffer (~50 ul).
Prepare a labelled 1.5 mL microcentrifuge containing 500 µL BLP digest buffer. Transfer the biopsies into the buffer and cut up with micro-scissors.
Pipette gently 5 times with 1 mL pipette to break up the tissue, ensuring all tissue pieces pass into pipette tip. Pipette gently 5 times with 1 mL pipette every ten minutes, making sure to pass all tissue pieces through pipette tip, return to ice.
At 30 min, add 200 µL 25% BSA to overwhelm the enzyme, pipette gently 5 times with 1 mL pipette to break up remaining chunks.
Top up tube to 10 mL using ice cold HBSS-/- buffer.
Centrifuge at 350 g @4°C for 5 min. Pour off supernatant gently (flick the last drop out) and re-suspend by gentle pipetting with 1 mL pipette in residual buffer (~50 µL).
..
Note
The small cell pellet is loose, so do not be tempted to remove supernatant with a pipette tip.

Add 500 µL Collagenase IV + DNAse I digest buffer and pipette gently 5 times with 1 mL pipette.
Incubate @ ~20°C (room temp): 10 mins for terminal ileum (TI), 15 mins for rectum (R).
Add 5 µL of 0.5M EDTA to quench collagenase IV and pipette gently 5 times with 1 mL pipette.
Pass digested cell suspension over 30 µm strainer into a new 15 mL tube, pour wash buffer through strainer into tube to 10 mL. Discard strainer.

Note
Avoid Flomi strainers, which seem to damage the epithelial cells.

Centrifuge at 350 g @4°C for 5 min.
Pour off the supernatant (flick the last drop out!). Let the tube stand for a minute, then gently resuspend cells with 1 mL pipette in the residual buffer (~50 µL)
Add 1 mL ACK lysis buffer, pipette gently 2 times with 1 mL pipette, and incubate 3 min at room temperature.
Add 8-10 mL wash buffer, centrifuge 350 g @4°C for 5 min, pour off supernatant, resuspend in residual buffer. Repeat this step for a total of 2 washes. After the second wash make sure to allow all wash buffer to drip off, flicking occasionally. Return tube to ice, approx 50-80 µL residual buffer will pool after 1-2 mins. [NB: occasionally there seems to be no visible pellet after ACK step.]
Gently resuspend cells using 1 mL pipette and pass over a 40 µm strainer held over a 6 well plate. Use 1 mL pipette to collect strained cell suspension from underneath the strainer.
Count cells using trypan blue: combine 10 µL trypan blue and 10 µL of cells and transfer 10 µL to a hemocytometer and count using a light microscope. Average quadrant count *20 = cell count per µL.
Take images for QC of cells. Quality target: Cell viability >70% and little/no observable cell debris.
Cell suspension QC:
AB
Issue: Repeat following step:
Cell clumps Pipette and 40 µm filter
Debris Wash and 40 µm filter
Dead cells ACK lysis, 2x wash and 40 µm filter

Note
Be aware that with each wash the cell count will decrease!

For 10X Chromium single cell gene expression protocol:
-Dedicated RNase-free pipettes and tips recommended.
-Adjust cell count to ~600-1000 cells/µL with wash buffer if necessary.
-Retrieve kit parts in the recommended temperatures 30 minutes before finishing.

Micro-scissors can be cleaned with Virkon (immerse 10 mins), wiped on Azo-wipe laid flat on a surface (do not hold the Azo-wipe), air-dried and re-used.

Cryopreservation
If immediate processing of the samples is not feasible, add biopsies to cold cryo buffer (90% HI-FBS:10% DMSO) within an hour of colleciton, and immediately store cryo tubes in Mr Frosty or Corning Coolcell at -80oC over night.
Transfer to liquid N2 the next day. If liquid N2 is not available at site, you can still get useable scRNAseq data from biopsies stored at -80oC for up to 4 weeks.
On the day of processing, thaw quickly, wash 2 times with cold 5% heat inactivated FBS in HBSS-/- and continue processing as for fresh.