Sep 15, 2020
Version 2
Nuclei isolation from human intestinal biopsic tissue for single-cell genomic applications V.2
- 1University of Chicago
- Human Cell Atlas Method Development Community
- Helmsley project_Basu lab
Protocol Citation: Ran RZ Zhou, Oni Basu 2020. Nuclei isolation from human intestinal biopsic tissue for single-cell genomic applications. protocols.io https://dx.doi.org/10.17504/protocols.io.bma2k2geVersion created by Ran RZ Zhou
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: In development
We are still developing and optimizing this protocol
Created: September 11, 2020
Last Modified: September 15, 2020
Protocol Integer ID: 42042
Keywords: gut, intestine, human, nuclei, single cell,
Disclaimer
The lysis buffer is formulated from the recipe in:
Drokhlyansky E, Smillie CS, Van Wittenberghe N, et al. The Human and Mouse Enteric Nervous System at Single-Cell Resolution [published online ahead of print, 2020 Aug 21].Cell. 2020;S0092-8674(20)30994-6. doi:10.1016/j.cell.2020.08.003
Abstract
This protocol provides an efficient method to isolate nuclei from human intestinal biopsy samples for single cell applications (RNA-seq or ATAC-seq).
Guidelines
The human intestinal tissue were obtained with patient consent and approval by the Institutional Review Board at the University of Chicago (IRB Number: 15573A). All the samples are processed for research use only.
Materials
MATERIALS
5M Sodium Chloride, 1000mlPromegaCatalog #V4221
BSASigma AldrichCatalog ##A8806
RiboLock RNase Inhibitor (40 U/µL)Thermo FisherCatalog #EO0381
0.5M EDTAFisher ScientificCatalog #2482-500
10 x PBS no calsium no magnesiusmFisher ScientificCatalog #BP399500
UltraPure™ DNase/RNase-Free Distilled WaterThermofisherCatalog #10977023
Red blood cell lysis buffer 10x Miltenyi BiotecCatalog #130-094-183
Tween 20Sigma AldrichCatalog #P7949
1M Tris-HCl pH 7.5Thermo Fisher ScientificCatalog #15567027
1M CaCl2Sigma-aldrichCatalog #21115
1M MgCl2Sigma-aldrichCatalog #63069
Lysis buffer 10 ml (make fresh)
5 ml 2x ST buffer
300 ul 1% Tween-20
50 ul 2%BSA
10 ul RNAse Inhibitor stock
4.64 ml UltraPure water
2x ST buffer 10 ml (Store at 4 Celsius up to 1 month)
292 mM NaCl
20 mM Tris-HCl pH 7.5
2 mM CaCl2
42 mM MgCl2
Bring up to volume with UltraPure water
RBC lysis buffer 10 ml
1 ml Red blood cells lysis buffer 10x
9 ml ultra pure water
2% BSA 10 ml (Store at 4 Celsius up to 1 month)
0.2 g BSA
10 ml UltraPure water
1% Tween-20 10 ml (Store up to 1 month)
1ml 10% Tween-20
9 ml UltraPure water
Nuclei suspension buffer 10 ml (make fresh)
10 ul RNAse Inhibitor stock
50 ul 2% BSA
9.94 ml 1x PBS
1x PBS 500 ml (filter through 0.2 uM filter top)
50 ml 10x PBS
450 ml UltraPure water
Sample preparation
Sample preparation
Rinse fresh samples in ice-cold PBS twice.
Note
Biopsy tissuse can be store up to 3 days in liquid nitrogen/at - 80 Celsius following the steps below:
Tissues are rinsed in ice-cold PBS twice
Flash freeze the tissue in 1.7 ml Eppendorf tube in liquid nitrogen
Store frozen tissue in liquid nitrogen (preferred) or at -80C
Start from step 2 if working with frozen tissues.
Tissue lysis
Tissue lysis
Mince the tissue, with 200 ul lysis buffer added, in a 1.7 ml Eppendorf tube by Iris Scissors on ice x 1 mins.
Add 1-1.5 ml ice-cold lysis buffer to the tube and incubate on ice x 5 mins. Invert the tube 3 times in the middle of the incubation to mix.
Wet a 40 micron cell strainer with 1 ml lysis buffer.
Filter the lysis through the strainer. Wash the strainer by 3 ml lysis buffer and 4 ml nuclei suspension buffer (NSB). Keep the flow through as this is where your nuclei are.
Nuclei collection
Nuclei collection
Spin down the flow-through, at 600 g x 5 mins, at 4 celsius in a 15 ml conical tube.
600 x g, 4°C, 00:05:00
Suspend nuclei in 100 ul NSB using gentle pipetting.
Note
If working with a single-cell platform, e.g. 10x Genomics, the nuclei should be suspended in PBS + 1% BSA + 0.2 U/ul RNase Inhibitor
If red bloold cells are present in fresh tissue nuclei suspension, dilute the suspension with NSB to 1 ml. Add 2 ml RBC lysis buffer and incubate on ice for 5 minutes. Pellet nuclei by centrifugation 600 g x 5 mins, 4 Celsius. Suspend nuclei in 100 ul NSB with gentle pipetting.
600 x g, 4°C, 00:05:00
Take 10 ul nuclei suspension and mix with 10 ul DAPI or Hoechst dye at 10 ug/ml and 10 ul WGA dye at 1 ug/ml. Count the nuclei.
DAPI/Hoechst staining-blue; WGA staining-orange Intact nuclei are co-stained by DAPI/Hoechst and WGA.
Nuclei preparation
Nuclei preparation
Dilute nuclei to the desired density using NSB.