This post is to troubleshoot coral nuclei isolations for single cell ATAC-seq. I will be troubleshooting protocols from multiple papers and on a few species.

Trial 1

This trial is based directly from Roquis et al. 2022.

I am also going to think about preserving the nuclei using this protocol

Materials

Equipment:

• Cold centrifuge with 15mL rotor
• Air Brush
• 15mL tubes
• Sterile whirlpak
• Dounce homogenizer

Chemicals:

• KCl
• NaCl
• MgCl₂
• Tris/Cl pH 7.4
• Sucrose
• Sodium buryrate
• Dithiotreitol (DTT)
• Phenylmethanesulfonyl fluoride (PMSF)
• cOmplete protease inhibitor (Roche cat #11836145001)
• NP-40
• CaCl₂
• RNAse inhibitor

Methods

Make buffers

Table 1: Chemical concentrations of each buffer. I will break down the volumes in the following tables but this is from the paper.

	Buffer 1	Buffer 2	Buffer 3	MNase Buffer
KCl	30 mM	30 mM	30 mM
NaCl	500 mM	500 mM	500 mM	500 mM
MgCl2	2.5 mM	2.5 mM	2.5 mM	40 mM
Tris/Cl pH 7.4	15 mM	15 mM	15 mM	10 mM
Sucrose	1 M	1 M	1.5 M	1 M
Sodium buryrate	5 mM	5 mM	5 mM	5 mM
Dithiotreitol (DTT)	25 mM	25 mM	25 mM
Phenylmethanesulfonyl fluoride (PMSF)	0.5 mM	0.5 mM	0.5 mM	0.1 mM
cOmplete protease inhibitor	1 X	1 X	1 X
NP-40		0.3 %
CaCl2				10 mM

Tissue dissociation (Adapted from Grace Snyder)

• Mechanical Dissociation:
  • Remove cells/tissue from skeleton with air brush and cold cell media
  • “Cell media” refers to 3.3X PBS (salinity ~ 35 ppt; Mg- and Ca-free), 2% FBS, 20 mM Hepes, pH 7.4
  • Mechanical dissociation is preferred due to shorter preparation time, but enzymatic dissociations are also optional; best for coral are dispase and liberase (~45 minutes for 1 cm2 fragment of a bouldering coral)
• Cell filtration:
  • filter tissue slurry collection through 40um cell strainer (P. damicornis).
  • For organisms with thicker mucus secretions, use a larger pore strainer first, such as 70 or 100um, followed by the 40um. Keep on ice.
• Optional:
  • dose with appropriate volume of RNAse inhibitor (Millepore Sigma Protector RNAse Inhibitor)
  • 1 uL or 40 units per 1 mL of cell slurry

Nuclei isolation

• Centrifuge tubes at 800 g at 4°C for 10 minutes.
• Carefully remove the supernatant and gently resuspend the pellets in 1 mL of buffer 1 and 1 mL of buffer 2.
• Transferred to a Dounce homogenizer and grind on ice for 4 minutes with pestle A, and let it rest on ice for 7 minutes.
• Transfer liquid to corex tubes containing 8 mL of buffer 3, in a manner that the homogenate would form a layer on top of buffer 3.
  • The two solutions have a different density, which allow them to stay one on top of the other without mixing.
• Centrifuge for 20 mins at 4°C and 7,800 g, with the lowest break speed possible.
  • This step allows separating coral nuclei, which will form a pellet at the bottom of the tube, from cell debris and Symbiodiniaceae cells, which stay at the interphase between the two buffers.
• Completely remove the supernatant by pouring out of the tubes and then by aspiration with micropipette.
• The pellets now contain coral nuclei and chromatin.

Quality Control

Preliminary protocol taken from Nadelmann et al. 2021.

• FACS

  • Proceed to FACS (which should occur at least fifteen minutes after staining with NucBlue).
  • Follow instructions for flow activated cell sorting to separate Hoechst-positive nuclei from small-particle debris
  • Sort through the entire sample to maximize the yield of nuclei.
  • Each sample should run through the FACS machine for approximately thirty minutes

• Microscopy

  • A standard light microscope can be used with a 10x or 20x lens to view the nuclei.
  • Microscopes with automated platforms, such as a Keyence, can also be used, to observe the presence of the fluorescent DAPI label in the nuclei, as the sample had been previously stained with NucBlue.
  • Check nuclei morphology to confirm presence of nuclei and removal of debris. An intact nucleus should be round and 8–12 μm in diameter (e.g. nuclei from heart tissue)

• RNA Extraction

  • Sion from the UM Oncogenomics core suggested to perform an RNA extraction of some of the isolated nuclei to ensure we have high quality RNA.
  • We should also sequence a few too.