Mar 07, 2020

Public workspaceProtocol for nuclear extraction from human heart tissue for single cell sequencing

DOI
dx.doi.org/10.17504/protocols.io.bcjqiumw
  • 1University of Washington
  • Human Cell Atlas Method Development Community
Christian Pfleger
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DOI: dx.doi.org/10.17504/protocols.io.bcjqiumw
Protocol Citation: Christian Pfleger, Shin Lin 2020. Protocol for nuclear extraction from human heart tissue for single cell sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.bcjqiumw
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: February 14, 2020
Last Modified: March 07, 2020
Protocol Integer ID: 33104
Keywords: nuclear extraction, human heart tissue, heart tissue, single cell sequencing,
Abstract
This protocol is for nuclear extraction from human heart tissue for single cell sequencing.
Attachments
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Materials
Required Solutions and Reagents


DAPI


Methanol (100 % )


Stock cell lysis buffer (store at Temperature4 °C ): 10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2


Recipe for 1 ml Cell lysis buffer - prepare fresh - 10 ml/sample required
  • Amount950 µL stock cell lysis Buffer
  • Amount10 µL IGEPAL CA-630
  • Amount10 µL 20 U/µl SUPERase•In RNase Inhibitor
  • Amount10 µL 10 % BSA
  • Amount10 µL 0.2 M Spermine
  • Amount10 µL 10 % Tween-20


(A) OptiPrep (product stock)


(B) OptiPrep diluent (store at Temperature4 °C ): 150 mM KCl, 30 mM MgCl2, 120 mM Tris-HCl (pH7.4)


(C) Working solution - prepare fresh - 50 % iodixanol - 13.5 ml/sample required
  • Amount11.25 mL Optiprep (A)
  • Amount2.25 mL Optiprep diluent (B)
  • Amount135 µL 20 U/µl SUPERase•In RNase Inhibitor
  • Amount135 µL 10 % BSA
  • Amount135 µL 0.2 M Spermine


Stock homogenization buffer: 0.25 M Sucrose, 25 mM KCl, 5 mM MgCl2, 20 mM Tris-HCl


(D) Homogenization buffer - prepare fresh - 6 ml/sample required
  • Amount970 µL stock homogenization buffer
  • Amount10 µL 20 Uµl SUPERase In RNase Inhibitor
  • Amount10 µL 10 % BSA
  • Amount10 µL 0.2 M Spermine


Recipe for 1 ml of Nuclear buffer - prepare fresh - 4 ml/sample required
  • Amount940 µL stock homogenization buffer
  • Amount10 µL 20 U/µl SUPERase•In RNase Inhibitor
  • Amount10 µL 10 % BSA
  • Amount10 µL 0.2 M Spermine
  • Amount10 µL 10 % Tween-20


Gradient Solutions
Working Solution (C) / mlHomogenization buffer (C) / mlper sample
30 % Optiprep10.61.6
35 % Optiprep7310
40 % Optirprep415
per solution124.6

Safety warnings
Please see SDS (Safety Data Sheet) for hazards and safety warnings.
Before start
Note: Be organized, diligent and keep sample and solutions cold at all times

Prepare required solutions and buffers fresh.
On dry ice
On dry ice
Put on dry ice:
  • flat bottom mortar and pestle, hammer and foreceps
  • sample-flash frozen heart tissue
  • scale plate
Once everything is cold
Once everything is cold
Assemble scale and cover plate with weighing paper.
Weigh Amount300 mg tissue.
Transfer tissue immediately into mortar and cover with pestle.
In laminar air hood – on dry ice
In laminar air hood – on dry ice
Pulverize tissue in mortar using pestle and hammer.
Hammer gently, scrape off tissue stuck to pistill.
Hammer again 3-6x.
In laminar air hood – on wet ice
In laminar air hood – on wet ice
Transfer pulversized tissue in 6 cm dish containing Amount4 mL cell lysis buffer TemperatureOn ice .
Start timer.
Segregate particles and transfer into douncer A with transfer pipette.
Wash plate with Amount2 mL cell lysis buffer and transfer into douncer A .
Dounce carefully 30x.
Filter through 100 µm mesh in 50 ml Falcon tube.
Wash douncer A with Amount2 mL cell lysis buffer and filter as well.
Keep Amount10 µL for QC #1.
Transfer into douncer B.
Dounce 20x.
Filter through 40 µm mesh in 50 ml Falcon tube.
Wash douncer B with Amount2 mL cell lysis buffer and filter as well.
Transfer into 15 ml Falcon tube.
Take time: should take Duration00:10:00 .
Spin Centrifigation400 x g, 4°C, 00:07:00 .

Aspirate supernatant.
Centrifugation
Centrifugation

Note
During testing, collect all 3 phases of Optiprep centrifugation, add same volume of nuclear buffer and spin to check for quality and quantity of seperation of nuclei and cell debris. Adjustments may be required.


Resupend pellet in Amount600 µL homogenization buffer (D) .
Add Amount1 mL Optiprep working solution and mix carefully (C) - 30 % iodixanol.
Keep Amount10 µL for QC #2.
Transfer into centrifugation tube (40ml).
Underlayer carefully nuclear sample with Amount8 mL Concentration35 % iodixanol using serological pipette.
Underlayer carefully both layers with Amount4 mL Concentration40 % iodixanol .
Centrifuge at Centrifigation8.000 x g, 4°C, 00:20:00 ; no breaks.
Collect ring of nuclei at 35 % - 40 % iodixanol interface.
Add same volume of nuclear buffer.
Spin at Centrifigation500 x g, 4°C, 00:10:00 .

Aspirate carefully and resuspend in nuclear buffer.
Stain Amount5 µL of sample as well as all fractions of QC with DAPI.
Check nuclei for complete lysis, nuclei morphology, purity and count.
Fixation
Fixation
Resuspend nuclei in Amount100 µL nucelar buffer .

Add drop wise Amount400 µL Concentration100 % (Temperature-20 °C ) methanol to suspension and transfer into Temperature-80 °C .