Protocol for nuclei isolation from fresh and frozen tissues using Salty-Ez10 buffer: compatible with snRNA-Seq and Multiome workflows from 10x Genomics

Luciano G Martelotto

Published: 2021-08-08 DOI: 10.17504/protocols.io.bw6qphdw

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Abstract

This is a protocol in development, which means it has not yet tested/challenged with multiple samples. So, please make sure you take it for a test drive before committing to it. Once you do please share your experience with me via email, Twitter or as a comment.

In this version I labelled DTT as optional since after further testing I have not seen any difference with or without in snRNA-Seq workflow. Also, I recommend WRB1 for snRNA-Seq workflow only (although still works fine for Multiome) and WRB2 for both snRNA-Seq and Multiome workflows. Since WRB2 is 10x Genomics' recommendation for Multiome I have adopted this for this workflow.

I include a discussion about cycling during cDNA amp.

Before start

All samples and reagents are kept on ice or at 4 °C (wet ice).

Prepare all buffers and reagents as described in the "Materials" section.

Steps

Tissue Homogenization

Mince/chop tissue with a razor blade to small pieces. The tissue may be as small as a grain of rice.

Note

For mincing the tissue, you may take the tube out of ice, however, be quick and return it to ice.

Add 300µL of chilled Salty-Ez10 Lysis Buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL) to the tissue in 1.5 mL tube.

Gently homogenize the sample using a douncer by stroking 10-20 times. Keep nuclei suspension on ice at all times.

Add an extra 700µL of chilled Salty-Ez10 Lysis Buffer (supplemented with RNAse Inhibitor 0.2-0.5 U/uL), mix gently by pipetting using wide-bore tips and incubate on ice for 0h 5m 0s. Repeat mixing 2-3 times during the incubation.

Note

Lysis Optimization can be done using the Luna-FL™Dual Fluorescence Cell Counter (this is the best automatic counter to me and makes Countess look like a Random Number Generator!) and by taking aliquots at different times until you get less than 5% viability using Acridine Orange/PI dual staining.

Nuclei Isolation

Filter homogenate using a 70 μm-strainer mesh to fit a pre-cooled 15 ml Falcon tube (e.g. pluriStrainer Mini 70 μm Cell Strainer). This step is to remove undigested tissue or fat prior to centrifugation.

Transfer flow though into a 1.5 mL LoBind tube and centrifuge the nuclei at 500x g for 0h 5m 0s at 4°C (these 5’ count as lysis time too!). Remove supernatant leaving behind ~50µL if the pellet seems loose.

Optional: add 1mL of Salty-Ez10 Lysis Buffer (supplemented with RNAse Inhibitor 0.2-if pellet is loose0.5 U/uL), gently resuspend pellet (optional: incubate for 0h 5m 0s on ice for additional lysis). Then centrifuge the nuclei at 500x g for 0h 5m 0s at 4°C.

Nuclei Wash and Resuspension

After removing the supernatant, add 500µL of WRB1 (supplemented with RNAse Inhibitor 0.2-0.5 U/uL) without disturbing the pellet . Let sit for 5’ on ice and then gently resuspend the pellet (this incubation is important to avoid clumping).

--> ALTERNATIVELY, resuspend the pellet in WRB2 (supplemented with RNAse Inhibitor 0.2-0.5 U/uL). The 5’ incubation is optional for this buffer. WRB2 is my preferred choice when we don't use OptiPrep for cleaning up nucs (like in case of cell lines which produce very little debris, if any) because for some samples I have seen nucs clumping when resuspended directly in WRB1 directly after Salty-Ez10. If you do OptiPrep then either WRB is fine.

Note

Warning : some nucs preps clump when you add WRB1 (this is a known phenomenon with Tris to PBS enchange, hence 5’ on ice helps but not always! It’s sample dependent). In that case use WRB2. Both WRBs are compatible with all 10x kits.

Note

Important: for 10x Multiome (10x Genomics) add 0.01% Digitonin in the WRB buffers (supplemented with RNAse Inhibitor 0.2-0.5 U/uL and 0.1% Tween-20), not during lysis step itself. In this way the time the nuclei is leaky (due to holes caused by Digitonin) is shorter, 5’ on ice is enough to permeabilize nuclei. Then replace it by the 1x Nuclei Buffer (NB) as indicated in the user guide (this buffer is essential for ATAC or Multiome kit).

If cell debris and large clumps are observed, pass through a cell strainer. For low volume, use a 40 μm Flowmi Cell Strainer to minimize volume loss.

Note

If high amounts of debris or micelles (very round droplets of fat, see attached photo) is present you may perform OptiPrep® continuous gradient as done in DOF (If high amounts of debris or micelles (very round droplets of fat, see attached photo) is present you may perform OptiPrep® continuous gradient as done in DOF (dx.doi.org/10.17504/protocols.io.bs99nh96, steps 9-12). Use WRBs for washes and resuspension for snRNA-Seq. For Multiome use WRBs for washes and resuspension 1x NB for resuspension. Pass through a cell strainer (e.g., 40 μm Flowmi Cell Strainer). , steps 9-12). Use WRBs for washes and resuspension for snRNA-Seq. For Multiome use WRBs for washes and resuspension 1x NB for resuspension. Pass through a cell strainer (e.g., 40 μm Flowmi Cell Strainer).

10.

Check integrity and purity under microscope and count manually or using an automatic counter. For automatic counter I recommend Luna-FL™ Dual Fluorescence Cell Counter and Acridine Orange/Propidium Iodide (AO/PI) Cell Viability Kit. This instrument also provides information about size and multiplets which is very useful.

11.

Proceed to your amazing snRNA-Seq or Multiome experiment!

Note

Note on cycle number for cDNA amplification There is not a rule of thumb for what how many extra cycles one need to add to get sufficient material for library prep. 10x Genomics indicates that one could make a library with just 1 ng, which I tested and it does, but we have to ramp up the cycles during SI-PCR. However, the libraries are less complex (less UMI and genes per nucs) and saturation is reached early on. Arguably, I prefer to have an input of >20 ng when possible so for that I add at least 2-4 cycles to start with (note the range of cells input is quite wide for cell input, e.g. 200-6000 --> 12 cycles for 3' v3.1 and 2000-6000 --> 14 cycles for low RNA content cells in 5' v2). Data so far has been great with significantly complex libraries and not nearly as saturated in 3' v3.1 chemistry. For 5' v2 chemistry, we have used the same approach. It is indeed challenging to determine then number of cycles to use for samples of unknown cell composition. Since 10x Genomics kits do not come with enough reagents to run extra cycles in case you need to increase the yield, I rather do a few more cycles upfront and reduce the cycling at SI-PCR. If you have any suggestions or comment I am welcome to hear about it. Quoting 10x Genomics:"The optimal number of cycles is a trade-off between generating sufficient final mass for library construction and minimizing PCR amplification artifacts." Interesting, for Multiome kit the total number of cycles for the RNA workflow ends up being 4 cycles more than standard snNA-Seq alone, e.g. 7 cycles pre-amp + 9 cDNA amp (16) for 2000 nucs versus 12 for standard scRNA-Seq (whole cell recommendation) so it seems 4 cycles more is within the range 10x Genomics recommends.

Protocol for nuclei isolation from fresh and frozen tissues using Salty-Ez10 buffer: compatible with snRNA-Seq and Multiome workflows from 10x Genomics

Disclaimer

Abstract

Before start

Steps

Tissue Homogenization

Nuclei Isolation

Nuclei Wash and Resuspension

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