TITLE: A versatile nuclei extraction protocol for single cell multiome ATAC and gene expression in non model species 
AUTHORS: Rose Ruiz Daniels, Richard S Taylor, Ioannis Konstantinidis, Sarah Salisbury, Diego Perojil Morata, Jorge Manuel de Oliveira Fernandes, Emily Clark, Dan Macqueen, Diego Robledo

[DESCRIPTION]
Here we present a modified version of : dx.doi.org/10.17504/protocols.io.261genwm7g47/v2 that was used to successfully extract nuclei from an array of different tissue types for single cell sequencing and modify it with the purpose of extracting nuclei for single cell multiome ATAC and gene expression on the 10x chromium. The modifications for this protocol include: different concentration of RNase inhibitors, different quantities for nuclear isolation buffer, removal of unnecessary steps as well as QC specific for multiome analysis.
If you are looking to use this protocol for bulk ATAC-seq use of protease inhibitor cocktail PIC is recommended instead of RNase inhibitor on the snRNA-seq version of this protocol (dx.doi.org/10.17504/protocols.io.261genwm7g47/v2) please get in touch with the authors if you are unsure on how to do this.

[BEFORE_START]

Sampling and storage for nuclear isolation.
Animals must be appropriately euthanized and immediately processed. Approximately ~60 mg of  tissue is placed in one clearly labelled cryotube and immediately flash frozen in liquid nitrogen. This step is critical. The tissue must be preserved as fast as possible for optimal results. In the absence of liquid nitrogen, samples can be frozen in dry ice. Samples can be stored at -80 °C for up to a year prior to use. Older samples might still yield viable nuclei but this would need to be tested.


All reagents should be chilled on ice prior to use.
Samples should be kept frozen on dry ice until immediately before nuclei isolation, and all sample-handling steps should be performed on ice. 
The centrifuge should be pre chilled at 4 °C.

All reagents are given for 2 nuclear isolations.
Amounts of buffer especially those that contain RNase should be adjusted appropriately for each experiment prepared prior and RNase added immediately before use.  




[GUIDELINES]

Before using this prep for library preparation do a trial run.
It is recommended to conduct a trial, especially on a new tissue type, to adjust various parameters without introducing RNase. This allows for the adjustment of parameters such as mincing times, filter size, and dilution into the final buffer, which ultimately leads to the production of good quality nuclei.

For multiome snRNA-seq and single cell ATAC-seq, it is advisable to perform a trial run where the quality of the nuclei is assessed by examining their integrity and the quality of their nuclear membrane (as shown in the picture in step 2.1).












[STEPS]
SECTION: Nucleus isolation workflow for ST-based buffers
1. 
 
on ice, place a piece of frozen tissue into one well of a 6-well tissue culture plate with 1 mL TST. 

 

SECTION: Nucleus isolation workflow for ST-based buffers
2. on ice, mince tissue initially using Tungsten Carbide scissors for 30 s and then with Noyes Spring Scissors   for a total of 10 min.

 

SECTION: Nucleus isolation workflow for ST-based buffers
2.1. 5 min into the mincing gently pipette up and down with a p1000 pipette using a low retention filtered tip. The time in the dissociation buffer is critical. See image for how to assess the timing is correct by looking at your nuclei.
  

SECTION: Nucleus isolation workflow for ST-based buffers
3. Pass lysate through a   cell strainer .

SECTION: Nucleus isolation workflow for ST-based buffers
3.1. Add a further 1 mL of TST to the cell strainer immediately.
SECTION: Nucleus isolation workflow for ST-based buffers
3.2. Add 3 mL of freshly prepared ST buffer to the lysate. 

SECTION: Nucleus isolation workflow for ST-based buffers
3.3. Add the 5 mL of lysate to a marked 15 ml falcon tube (Corning) on ice.

SECTION: Nucleus isolation workflow for ST-based buffers
4. Centrifuge at 500 x g, 5 min, 4 °C in a swinging bucket centrifuge.

SECTION: Nucleus isolation workflow for ST-based buffers
5. Discard liquid, carefully remove excess liquid with a p200 pipette, careful to not disturve the pellet. Resuspend the pellet gently using a p1000 pipette in diluted nuclei buffer in aiming for target recover of 6.000 nuclei. The concentration of RNAse inhibitor should be 1U/ul.
 
7/v2

SECTION: Nucleus isolation workflow for ST-based buffers
6. Count the nuclei using a C-chip disposable haemocytometer. 
 

SECTION: Nucleus isolation workflow for ST-based buffers
7. The nuclei are also counted using a Bio-Rad TC20 to confirm results from the disposable haemocytometer and to count the proportion of viable cells.