Apr 07, 2020
Nuclei isolation from mouse lung for single nucleus RNASeq
- 1Division of Pulmonary and Critical Care, Department of Medicine, Washington University in St. Louis School of Medicine, USA;
- 2Division of Nephrology, Department of Medicine, Washington University in St. Louis School of Medicine, USA;
- 3Department of Developmental Biology, Washington University in St. Louis School of Medicine, USA
- Human Cell Atlas Method Development Community
Protocol Citation: Jeffrey Koenitzer, Ben Humphreys 2020. Nuclei isolation from mouse lung for single nucleus RNASeq. protocols.io https://dx.doi.org/10.17504/protocols.io.bdv2i68e
Manuscript citation:
Koenitzer, J. R., Wu, H., Atkinson, J. J., Brody, S. L., Humphreys, B. D. (2020). Single nucleus RNASeq profiling of mouse lung: reduced dissociation bias and improved detection of rare cell types compared with single cell RNASeq. bioRxiv. preprint doi: https://doi.org/10.1101/2020.03.06.981407
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: March 18, 2020
Last Modified: April 07, 2020
Protocol Integer ID: 34458
Keywords: nuclei isolation, mouse, mouse lung, RNASeq, single nucleus RNASeq,
Abstract
This protocol is for nuclei isolation from mouse lung for single nucleus RNASeq.
It is adapted directly from Joshi et al., with adjustments to RNase inhibitor concentrations and removal of FACS sorting steps.
CITATION
Materials
MATERIALS
RNaseZap®Thermo ScientificCatalog #AM9780
cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpackRocheCatalog #05 892 791 001
RNasin Plus Ribonuclease InhibitorsPromegaCatalog #N2615
SUPERaseIN RNase InhibitorThermo Fisher ScientificCatalog #AM2696
RNase free H2OThermo ScientificCatalog #AM9938
Albumin, Bovine Serum, 10% Aqueous Solution, Nuclease-FreeMillipore SigmaCatalog #126615-25ML
Gibco™ DPBS no calcium no magnesiumThermo Fisher ScientificCatalog #14190144
Nuclei Isolation Kit: Nuclei EZ PrepSigma AldrichCatalog #NUC-101
Note
The Nuclei Isolation Kit contains the Nuclei EZ Lysis Buffer that is required for the praparation of Lysis Buffer and cOmplete stock (10x).
Storage Conditions
| Material | Storage | |
| 1x DPBS | 4 ºC | |
| Nuclei EZ Lysis Buffer (from kit NUC-101) | 4 ºC | |
| cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack | 4 ºC | |
| RNasin Plus Ribonuclease Inhibitors | -20 °C | |
| SUPERaseIN RNase Inhibitor | -20 °C | |
| RNase free H2O | RT | |
| RNaseZap | RT | |
| Bovine serum albumin, 10% solution, nuclease free | -20 °C |
Equipment
gentleMACS™ C Tubes
NAME
Tissue dissociators and tubes
TYPE
gentleMACS™
BRAND
130-093-237
SKU
LINK
Equipment
gentleMACS™ Dissociator
NAME
Tissue Dissociator
TYPE
MACS
BRAND
130-093-235
SKU
LINK
Equipment
pluriStrainer® 40 µm
NAME
Cell Strainer
TYPE
pluriSelect
BRAND
43-50040
SKU
LINK
Equipment
pluriStrainer® 5 µm
NAME
Cell Strainer
TYPE
pluriSelect
BRAND
43-50005
SKU
LINK
Equipment
C-Chip™ Disposable Hemacytometers (Fuchs Rosenthal)
NAME
Counting Chamber
TYPE
INCYTO
BRAND
DHCF015
SKU
LINK
Equipment
Falcon™ 15mL Polystyrene Conical Centrifuge Tubes
NAME
Centrifuge Tubes
TYPE
Falcon™
BRAND
352095
SKU
LINK
Equipment
Ambion® RNase-free 50 ml Conical Tubes
NAME
Centrifuge Tubes
TYPE
Ambion®
BRAND
AM12502
SKU
LINK
Equipment
TPP 60 mm Tissue Culture Dishes
NAME
Tissue Culture Dish
TYPE
TPP
BRAND
TP93060
SKU
LINK
Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
Prepare Working Solutions
Complete stock (10x)
- 1 mL Nuclei EZ Prep lysis buffer
- 1 tablet cOmplete ULTRA tablets
Lysis Buffer - 2 ml per < 6 mm3 tissue
- 200 µL cOmplete stock
- 1.775 mL Nuclei EZ Prep lysis buffer
- 12.5 µL RNasin Plus
- 12.5 µL SUPERaseIN
Wash Buffer - 4 ml per < 6 mm3 tissue
- 3.575 mL dPBS
- 400 µL 10% BSA
- 12.5 µL RNasin Plus
- 12.5 µL SUPERaseIN
Resuspension Buffer – 1 ml
- 0.977 mL dPBS
- 10 µL 10% BSA
- 6.25 µL RNasin Plus
- 6.25 µL SUPERaseIN
Pre-cool all instruments (including centrifuges), buffers, and tubes.
Note
Note: All steps are performed On ice or in cold room (4 °C ) to minimize RNA degradation.
Remove mouse lung sample from -80 ºC freezer and trim a ~6 mm3 piece.
Thaw on small plastic weighing boat until able to insert 26G needle to tissue, then inject ice cold 1 mL Lysis Buffer to ‘inflate’ the tissue. Add the remaining Lysis Buffer and chop to the smallest pieces possible with scissors (60 s).
Transfer the minced tissue and buffer to a GentleMacs C tube.
Close, invert, and transfer directly to MACS Tissue dissociator.
Run m_lung_01 program and m_lung_02 program in sequence, stopping the latter after 00:00:20 .
Foam after GentleMacs
Place tube On ice .
Reduce foam by centrifugation (750 x g, 00:01:00 ).
Foam after centrifugation
Using a wide bore tip, pipette up and down to recover any pelleted material and pass lysate through 40 µm filter to 50 ml conical tube.
Wash strainer with 4 mL Wash Buffer .
Pass suspension through a 5 µm strainer into 50 ml conical tube.
Centrifuge at 600 x g, 4°C, 00:05:00 .
Resuspend in 500 µL Resuspension Buffer .
Count nuclei by hemocytometer and dilute to desired concentration (e.g. 10,000 per µl).
Note
Note: it is easier to resuspend in lower volumes and dilute than to concentrate nuclei via further centrifugation.
40x Nuclear suspension with scant debris
Proceed to 10x Chromium.
Citations
Nikita Joshi, Alexander Misharin. Single-nucleus isolation from frozen human lung tissue for single-nucleus RNA-seq
dx.doi.org/10.17504/protocols.io.zu8f6zw