Sep 15, 2020
Intestinal Organoid Dissociation and Nuclei Isolation for Single Cell ATAC-Seq
- 1University of Chicago
- Human Cell Atlas Method Development Community
- Helmsley project_Basu lab
Protocol Citation: Heather Eckart, Ran RZ Zhou, Nadia Khan 2020. Intestinal Organoid Dissociation and Nuclei Isolation for Single Cell ATAC-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bmdbk22n
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: September 15, 2020
Last Modified: September 15, 2020
Protocol Integer ID: 42115
Abstract
This protocol provides a procedure for human intestinal organoid dissociation into a single cell suspension and nuclei isolation prior to Single Cell ATAC-Sequencing.
Guidelines
Nuclei isolation for Chromium Next GEM Single Cell ATAC Sequencing was performed following the protocol provided by 10X Genomics. For further guidelines and tips reference the original protocol below.
Chromium Next GEM Single Cell ATAC Sequencing was performed following the protocol provided in the user guide from 10X Genomics.
The primary human tissue that generates the organoids, are obtained from endoscopic biopsies after patient's consent and approval from Institutional Review Board at the University of Chicago (IRB Number: 15573A).
Materials
MATERIALS
Magnesium chloride solution for molecular biology (1.00 M)Sigma – AldrichCatalog #M1028
TrypLE™ Express Enzyme (1X), no phenol redThermo FisherCatalog #12604013
Wheat Germ Agglutinin, Alexa Fluor™ 594 ConjugateThermo FisherCatalog #W11262
Trizma Hydrochloride Solution pH 7.4 Sigma AldrichCatalog #T2194
Sodium Chloride Solution 5 M Sigma AldrichCatalog #59222C
Nonidet P40 SubstituteSigma AldrichCatalog # 74385
MACS BSA Stock SolutionMiltenyi BiotecCatalog # 130-091-376
Flowmi Cell Strainer 40 µm Bel-ArtCatalog #H13680-0040
Nuclei Buffer 20X 10x GenomicsCatalog #2000153/2000207
DAPISigma AldrichCatalog #D9542
Note: 10x Genomics Nuclei Buffer 20X (2000153/2000207) is included in the 10x Genomics Single Cell ATAC Library Kits
Diluted Nuclei Buffer 1mL
Nuclei Buffer (20X) 50 ul (final concentration 1X)
Nuclease free water 950 ul
Safety warnings
Note: 10x Genomics Nuclei Buffer 20X (2000153/2000207) is included in the 10x Genomics Single Cell ATAC Library Kits
Diluted Nuclei Buffer 1mL
Nuclei Buffer (20X) 50 ul (final concentration 1X)
Nuclease free water 950 ul
Before start
Prepare diluted nuclei buffer, wash buffer, lysis buffer, and PBS with 0.04% BSA
Organoid Dissociation
Organoid Dissociation
Incubate organoid in TrypLE for up to 20 minutes at 37°C .
Every 5 minutes, pipette the cell suspension up and down 5-10 x and check the digestion progress with a hemocytometer until over 90% of the cells are singlet cells.
Nuclei Isolation
Nuclei Isolation
For freshly dissociated cells, perform 1-2 washes with PBS + 0.04% BSA (20ul BSA/1mL 1X PBS).
Note
Consult 10X Genomics protocol for using frozen cells.
Determine the cell count after washing using a hemocytometer.
Add cell suspension of 100,000-1,000,000 cells to a 2-ml tube. For our experiment we started with 200,000 cells per sample.
Centrifuge at 300 rcf for 5 min at 4°C.
300 x g, 4°C, 00:05:00
Remove ALL the supernatant without disrupting the cell pellet.
Add 100 µl chilled Lysis Buffer. Pipette to mix 10x.
100 µL Lysis Buffer
Incubate for 4 min on ice.
On ice 4 min
Note
Time may vary depending on cell type; 4 minutes is specific for organoid samples.
Add 1 ml chilled Wash Buffer to the lysed cells. Pipette to mix 5x.
1 mL Wash Buffer
Centrifuge at 500 rcf for 5 min at 4°C.
500 x g, 4°C, 00:05:00
Remove the supernatant without disrupting the nuclei pellet.
Based on your targeted nuclei recovery, cell concentration in step 4 and assuming ~50% nuclei loss during cell lysis, resuspend in chilled Diluted Nuclei Buffer (1x). Maintain on ice.
(See Nuclei Stock Concentration Table and Example Calculation below)
For our experiment, we targeted 5,000 nuclei.
Check nuclei integrity by staining with WGA and DAPI. Also determine the nuclei concentration using a hemocytometer
OPTIONAL: If cell debris and large clumps are observed, pass through a cell strainer. For low volume, use a 40 µm Flowmi Cell Strainer to minimize volume loss
Proceed immediately to Chromium Next GEM Single Cell ATAC Sequencing protocol (found in the Chromium Single Cell ATAC Solution User Guide)