TITLE: Protocol for nuclear extraction from human heart tissue for single cell sequencing
AUTHORS: Christian Pfleger, Shin Lin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for nuclear extraction from human heart tissue for single cell sequencing.</div></div>

[STEPS]
?. [On dry ice]
Put on dry ice:flat bottom mortar and pestle, hammer and foreceps sample-flash frozen heart tissue scale plate
?. [In laminar air hood – on dry ice]
Pulverize tissue in mortar using pestle and hammer.
?. [In laminar air hood – on wet ice]
Transfer pulversized tissue in 6 cm dish containing .
[cell lysis buffer]
on ice
?. [Once everything is cold]
Assemble scale and cover plate with weighing paper.
?. [Once everything is cold]
Weigh  tissue.
300 mg
?. [Once everything is cold]
Transfer tissue immediately into mortar and cover with pestle.
?. [In laminar air hood – on dry ice]
Hammer gently, scrape off tissue stuck to pistill.
?. [In laminar air hood – on wet ice]
Start timer.
?. [In laminar air hood – on dry ice]
Hammer again 3-6x.
?. [In laminar air hood – on wet ice]
Segregate particles and transfer into douncer A with transfer pipette.
?. [In laminar air hood – on wet ice]
Wash plate with   and transfer into douncer A .
[cell lysis buffer]
?. [In laminar air hood – on wet ice]
Dounce carefully 30x.
?. [In laminar air hood – on wet ice]
Filter through 100 µm mesh in 50 ml Falcon tube.
?. [In laminar air hood – on wet ice]
Wash douncer A with  and filter as well.
[cell lysis buffer]
?. [In laminar air hood – on wet ice]
Keep  for QC #1.
10 µl
?. [In laminar air hood – on wet ice]
Transfer into douncer B.
?. [In laminar air hood – on wet ice]
Dounce 20x.
?. [In laminar air hood – on wet ice]
Filter through 40 µm mesh in 50 ml Falcon tube.
?. [In laminar air hood – on wet ice]
Wash douncer B with  and filter as well.
[cell lysis buffer]
?. [In laminar air hood – on wet ice]
Transfer into 15 ml Falcon tube.
?. [In laminar air hood – on wet ice]
Take time: should take  .
?. [In laminar air hood – on wet ice]
Spin .
Centrifuge: 400 34, 7 min, 4 10
?. [In laminar air hood – on wet ice]
Aspirate supernatant.
?. [Centrifugation]
Resupend pellet in .
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.
[homogenization buffer (D) ]
?. [Centrifugation]
Add   - 30 % iodixanol.
[Optiprep working solution and mix carefully (C)]
?. [Centrifugation]
Keep  for QC #2.
10 µl
?. [Centrifugation]
Transfer into centrifugation tube (40ml).
?. [Centrifugation]
Underlayer carefully nuclear sample with  using serological pipette.
8 ml
[ iodixanol]
?. [Centrifugation]
Underlayer carefully both layers with  .
4 ml
[iodixanol]
?. [Centrifugation]
Centrifuge at ; no breaks.
Centrifuge: 8.000 34, 20 min, 4 10
?. [Centrifugation]
Collect ring of nuclei at 35 % - 40 % iodixanol interface.
?. [Centrifugation]
Add same volume of nuclear buffer.
?. [Centrifugation]
Spin at .
Centrifuge: 500 34, 10 min, 4 10
?. [Centrifugation]
Aspirate carefully and resuspend in nuclear buffer.
?. [Centrifugation]
Stain  of sample as well as all fractions of QC with DAPI.
5 µl
?. [Centrifugation]
Check nuclei for complete lysis, nuclei morphology, purity and count.
?. [Fixation]
Resuspend nuclei in .
[nucelar buffer]
?. [Fixation]
Add drop wise   () methanol to suspension and transfer into .
400 µl
-20 °C
-80 °C