Protocol for nuclear extraction from human heart tissue for single cell sequencing
Mar 07, 2020

Protocol for nuclear extraction from human heart tissue for single cell sequencing
In 1 collection

  • 1University of Washington
Abstract
This protocol is for nuclear extraction from human heart tissue for single cell sequencing.
Protocol Citation
Christian Pfleger, Shin Lin 2020. Protocol for nuclear extraction from human heart tissue for single cell sequencing. protocols.iohttps://dx.doi.org/10.17504/protocols.io.bcjqiumw
Keywords
nuclear extraction, human heart tissue, heart tissue, single cell sequencing
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
Created
Feb 14, 2020
Last Modified
Mar 07, 2020
Ownership history
  • Feb 14, 2020
    Julia Rossmanith
    protocols.io
  • Mar 06, 2020
    Christian Pfleger
PROTOCOL integer ID
33104
Parent protocols
Part of collection
2020 Featured Protocols
MATERIALS TEXT
Required Solutions and Reagents


DAPI


Methanol (100 % )


Stock cell lysis buffer (store at 4 °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
  • 950 µL stock cell lysis Buffer
  • 10 µL IGEPAL CA-630
  • 10 µL 20 U/µl SUPERase•In RNase Inhibitor
  • 10 µL 10 % BSA
  • 10 µL 0.2 M Spermine
  • 10 µL 10 % Tween-20


(A) OptiPrep (product stock)


(B) OptiPrep diluent (store at 4 °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
  • 11.25 mL Optiprep (A)
  • 2.25 mL Optiprep diluent (B)
  • 135 µL 20 U/µl SUPERase•In RNase Inhibitor
  • 135 µL 10 % BSA
  • 135 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
  • 970 µL stock homogenization buffer
  • 10 µL 20 Uµl SUPERase In RNase Inhibitor
  • 10 µL 10 % BSA
  • 10 µL 0.2 M Spermine


Recipe for 1 ml of Nuclear buffer - prepare fresh - 4 ml/sample required
  • 940 µL stock homogenization buffer
  • 10 µL 20 U/µl SUPERase•In RNase Inhibitor
  • 10 µL 10 % BSA
  • 10 µL 0.2 M Spermine
  • 10 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.
Note: Be organized, diligent and keep sample and solutions cold at all times

Prepare required solutions and buffers fresh.

On dry ice

1
Put on dry ice:
  • flat bottom mortar and pestle, hammer and foreceps
  • sample-flash frozen heart tissue
  • scale plate

Once everything is cold

2
Assemble scale and cover plate with weighing paper.
3
Weigh 300 mg tissue.
4
Transfer tissue immediately into mortar and cover with pestle.

In laminar air hood – on dry ice

5
Pulverize tissue in mortar using pestle and hammer.
6
Hammer gently, scrape off tissue stuck to pistill.
7
Hammer again 3-6x.

In laminar air hood – on wet ice

8
Transfer pulversized tissue in 6 cm dish containing 4 mL cell lysis buffer On ice .
9
Start timer.
10
Segregate particles and transfer into douncer A with transfer pipette.
11
Wash plate with 2 mL cell lysis buffer and transfer into douncer A .
12
Dounce carefully 30x.
13
Filter through 100 µm mesh in 50 ml Falcon tube.
14
Wash douncer A with 2 mL cell lysis buffer and filter as well.
15
Keep 10 µL for QC #1.
16
Transfer into douncer B.
17
Dounce 20x.
18
Filter through 40 µm mesh in 50 ml Falcon tube.
19
Wash douncer B with 2 mL cell lysis buffer and filter as well.
20
Transfer into 15 ml Falcon tube.
21
Take time: should take 00:10:00 .
22
Spin 400 x g, 4°C, 00:07:00 .

23
Aspirate supernatant.

Centrifugation

24

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 600 µL homogenization buffer (D) .
25
Add 1 mL Optiprep working solution and mix carefully (C) - 30 % iodixanol.
26
Keep 10 µL for QC #2.
27
Transfer into centrifugation tube (40ml).
28
Underlayer carefully nuclear sample with 8 mL 35 % iodixanol using serological pipette.
29
Underlayer carefully both layers with 4 mL 40 % iodixanol .
30
Centrifuge at 8.000 x g, 4°C, 00:20:00 ; no breaks.
31
Collect ring of nuclei at 35 % - 40 % iodixanol interface.
32
Add same volume of nuclear buffer.
33
Spin at 500 x g, 4°C, 00:10:00 .

34
Aspirate carefully and resuspend in nuclear buffer.
35
Stain 5 µL of sample as well as all fractions of QC with DAPI.
36
Check nuclei for complete lysis, nuclei morphology, purity and count.

Fixation

37
Resuspend nuclei in 100 µL nucelar buffer .

38
Add drop wise 400 µL 100 % (-20 °C ) methanol to suspension and transfer into -80 °C .