TITLE: Isolation of nuclei from frozen tissue for ATAC-seq and other epigenomic assays
AUTHORS: Ryan Corces, William J. Greenleaf, Howard Y. Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol enables the isolation of nuclei from frozen tissues. These nuclei are suitable for use in ATAC-seq, single-cell ATAC-seq, ChIP-seq, HiC/3C, and many other assays.</div></div>

[STEPS]
?. [Before you start the protocol:]
All steps should be performed on ice or at . Pre-chill a swinging bucket centrifuge and a fixed angle centrifuge to 4°C.
4 °C
?. [Before you start the protocol:]
Pre-chill all Dounces and pestles to  in a fridge.
4 °C
?. [Before you start the protocol:]
Pre-chill all tubes. For each sample you are processing, you will need: (i) One 2 ml round-bottom LoBind tube for gradient separation (ii) One 1.5 ml LoBind tube for RNA homogenate (iii) One 2 ml Nunc Cryotube for extra nuclei (iv) One 50 ml conical for filtration step (often optional)
?. [Before you start the protocol:]
Prepare all buffers. For faster dissolution, crush protease inhibitor tablets prior to addition to 1x Homogenization Buffer Unstable Solution. DTT, Spermidine, Spermine, and digitonin are stored at -20°C. All other detergents, ATAC-RSB, and other buffers are stored at . Do not prepare transposition mix ahead of time. a.	Remember that the catalog number provided for iodixanol from Sigma comes as a 60% solution (not 100%).
4 °C
?. [Before you start the protocol:]
Fill up a 2 L beaker with 500 ml sterile water to soak the used Dounces and pestles.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Remove samples from liquid nitrogen storage and keep on dry ice until use.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Place 20 mg frozen tissue into a pre-chilled 2 ml Dounce containing 1 ml cold 1x HB and let thaw for . For >30 mg tissue, use 2 ml 1x HB. For 10-20 mg tissue, use 1 ml 1x HB. For 50 um tissue sections, use 0.5 ml 1x HB.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
If you would like to collect RNA from the same sample, add 10 ul RiboLock per ml of 1x HB and mix well.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Dounce with “A” loose pestle until resistance goes away (~10 strokes).
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Place “A” pestle into beaker with sterile water to soak for cleaning later.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Dounce with “B” tight pestle for 20 strokes.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Place “B” pestle into beaker with sterile water to soak for cleaning later.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Filter during transfer using a 70 um Flowmi strainer and transfer homogenate to a pre-chilled 2 ml LoBind tube.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Place Dounce into beaker with sterile water to soak for cleaning later.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Pellet nuclei by spinning  at  at 350 RCF in a fixed angle centrifuge.
4 °C
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Remove all but 50 ul of supernatant (containing cytoplasmic RNAs) and transfer to a pre-chilled 1.5 ml LoBind tube. If the pellet is not clearly visible, you can leave more supernatant in the tube, up to 400 ul and add less of the 1x HB buffer in the next step.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Gently resuspend nuclei in a total volume of 400 ul 1x HB. If you only left 50 ul in the tube in the previous step, this means you should add 350 ul 1x HB. Make sure nuclei are fully resuspended without clumps.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Add 1 volume (400 ul) of 50% Iodixanol Solution and mix well by pipetting
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Slowly layer 600 ul of 30% Iodixanol solution under the 25% mixture. To avoid mixing of layers, wipe the side of the pipette tip with a Kimwipe to remove excess Iodixanol solution from the external surfaces of the pipette tip.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Layer 600 ul of 40% Iodixanol solution under the 30% mixture. To avoid mixing of layers, wipe the side of the pipette tip with a Kimwipe to remove excess Iodixanol solution from the external surfaces of the pipette tip.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
In a pre-chilled swinging bucket centrifuge, spin for  at  at 3,000 RCF with the brake off. Handle tubes gently so as to not disturb the gradient.
4 °C
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Using a vacuum, aspirate the top layers down to within 200-300 ul of the nuclei band at the 30%-40% interface. Be careful not to get too close as you will disrupt the nuclei band.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Using a 200 ul volume, collect the nuclei band and transfer to a fresh tube. Do not aspirate more than 200 ul at this step as this can cause you to take too much of the 40% layer which sometimes contains debris.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Dilute nuclei by adding some volume of ATAC-RSB-Tween Buffer. Mix gently by pipetting. The precise volume of ATAC-RSB-Tween to add will depend on how many nuclei you have. If you don’t dilute enough, it will be hard to get an accurate count. If you dilute too much, it will be similarly hard to get an accurate count. You should minimally add 200 ul of ATAC-RSB-Tween buffer to dilute the iodixanol as high concentrations of iodixanol can be too viscous for hemocytometers.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Optional – If residual un-homogenized tissue makes it difficult to Dounce, filter homogenate through a 70 um bucket-style cell strainer filter into a pre-chilled 50 ml conical prior to using tight pestle “B”.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
During this step, you will need to gradually draw your pipette tip up to avoid overflowing the tube. However, the tip of your pipette must stay below the 30%-40% interface at all times.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
Iodixanol is meant to be used at higher speeds (10,000 RCF) but high-speed swinging bucket centrifuges are not always readily available so we perform this step at 3,000 g and have not had any issues.
?. [Transposition of Nuclei]
Count nuclei using Trypan blue staining (1:1 ratio of Trypan to sample) and a manual hemocytometer. We recommend using disposable hemocytometers for consistency but do not recommend automated cell counters.
?. [Transposition of Nuclei]
We normally perform two technical replicates per sample. Each technical replicate should ideally have 50,000 nuclei, requiring 100,000 nuclei total. If you don’t have at least 100,000 nuclei, follow this convention: (A)	More than 50,000 nuclei, still do 2 technical replicates using half of the volume for each replicate but reduce the volume of Tn5 transposase proportionately to the number of nuclei. Maintain all other transposition reaction volumes. For example, for 25,000 cells, use 1.25 ul of Tn5 transposase in a 50 ul total reaction volume. Replace omitted Tn5 volume with water. (B) Fewer than 50,000 nuclei, only do 1 technical replicate and reduce the volume of Tn5 transposase proportionately to the number of nuclei. Maintain all other transposition reaction volumes. For example, for 25,000 cells, use 1.25 ul of Tn5 transposase in a 50 ul total reaction volume. Replace omitted Tn5 volume with water.
?. [Transposition of Nuclei]
Label and chill 1.5 ml LoBind tubes according to how many tubes will be needed for transpositions.
?. [Transposition of Nuclei]
Transfer 50,000 nuclei into a 1.5 ml LoBind tube containing 1000 ul of ATAC-RSB-Tween Buffer. If the total volume won’t fit in a 1.5 ml tube, just reduce the amount of ATAC-RSB-Tween that you add to the tube to start.
?. [Transposition of Nuclei]
Centrifuge nuclei for  at 500 RCF at  in a fixed angle centrifuge. At this point, the pellet should be clearly visible if 50,000 nuclei were used. Pellets of as few as 10,000 nuclei should be visible.
4 °C
?. [Transposition of Nuclei]
Using a p1000 pipette, remove all but the last 100 ul of supernatant. Remove last 100 ul with p200 pipette set to 200 ul using a single fluid pipetting motion. Place the tip of your pipette on the opposite side of the tube to where the nuclei pellet is located during this final aspiration step.
?. [Transposition of Nuclei]
Add 50 ul ATAC-seq Reaction Mix to each tube and pipette up and down 6 times to resuspend nuclei pellet.
?. [Transposition of Nuclei]
Incubate reactions at  for  in a thermoshaker with 1000 RPM constant shaking.
37 °C
?. [Transposition of Nuclei]
After incubation, add 250 ul (5 volumes) of Binding Buffer from the Zymo DNA Clean and Concentrator 5 kit. Mix well by vortexing and inverting to collect any condensate from the lid.
?. [Transposition of Nuclei]
Pulse centrifuge to collect volume in the bottom of the tube.
?. [Transposition of Nuclei]
Either finish the cleanup protocol using the Zymo DNA Clean and Concentrator 5 kit or transfer the binding buffer transposition mix to  for short term storage for up to 2 weeks.
-20 °C
?. [Transposition of Nuclei]
Unlike the published ATAC-seq protocols, you do not need to do an individual lysis step in this protocol because the nuclei are exposed to NP40 throughout the Douncing portion of the protocol.
?. [Transposition of Nuclei]
If you store the binding buffer transposition mix at , allow it to equilibrate to room temperature and mix well before proceeding with the Zymo DNA Clean and Concentrator clean up protocol. This will dissolve any precipitate that has formed and allow optimal binding to the column membrane.
-20 °C
?. [Cleanup and Freezing Down of Nuclei and Homogenate]
If you would like to save extra nuclei for other assays or to potentially use in additional ATAC-seq experiments downstream:
?. [Cleanup and Freezing Down of Nuclei and Homogenate]
If you would like to keep homogenate for making RNA (or potentially protein) downstream, store homogenate at .
-80 °C
?. [Cleanup and Freezing Down of Nuclei and Homogenate]
Cleaning Dounces and pestles: Rinse all Dounces and pestles thoroughly with sterile water (2x) followed by 70% ethanol (2x). Let Dounces and pestles dry on a kimwipe or paper towel for a few hours to overnight.
?. [Cleanup and Freezing Down of Nuclei and Homogenate]
Pellet remaining nuclei by centrifugation for  at 500 RCF at .
4 °C
?. [Cleanup and Freezing Down of Nuclei and Homogenate]
Carefully aspirate supernatant using two pipetting steps (p1000 then p200) as above.
?. [Cleanup and Freezing Down of Nuclei and Homogenate]
Gently resuspend nuclei pellet in 100 ul of cold BAM Banker media and transfer to a pre-chilled 2 ml Nunc cryovial.
?. [Cleanup and Freezing Down of Nuclei and Homogenate]
Slow-freeze nuclei in a freezing container and move to  or liquid nitrogen storage the next day.
-80 °C
?. [Processing Homogenate to Make RNA (optional)]
Pre-chill a fixed-angle centrifuge to .
4 °C
?. [Processing Homogenate to Make RNA (optional)]
Thaw homogenate on ice.
?. [Processing Homogenate to Make RNA (optional)]
Transfer 150 ul to a 2 ml LoBind tube containing 1500 ul Trizol and mix.
?. [Processing Homogenate to Make RNA (optional)]
Add 400 ul chloroform to the Trizol and mix by vortexing for .
?. [Processing Homogenate to Make RNA (optional)]
Immediately spin in a pre-chilled fixed-angle centrifuge at 21,000 RCF for  at .
4 °C
?. [Processing Homogenate to Make RNA (optional)]
Pipette clear aqueous layer (~650 ul) into a clean 1.5 ml LoBind tube.
?. [Processing Homogenate to Make RNA (optional)]
Add an equal volume (650 ul) of 100% ethanol and mix well.
?. [Processing Homogenate to Make RNA (optional)]
Pass all volume through a QIAgen RNeasy column using two centrifugation steps.
?. [Processing Homogenate to Make RNA (optional)]
Follow the QIAgen RNeasy protocol and elute in 27 ul of elution buffer
?. [Processing Homogenate to Make RNA (optional)]
Add 3 ul of 10x Turbo DNase Buffer and 1 ul of Turbo DNase enzyme.
?. [Processing Homogenate to Make RNA (optional)]
Incubate at  for .
37 °C
?. [Processing Homogenate to Make RNA (optional)]
Add 70 ul of RNase free water and 350 ul of QIAgen RLT Buffer and mix well.
?. [Processing Homogenate to Make RNA (optional)]
Add 250 ul 100% ethanol and mix well.
?. [Processing Homogenate to Make RNA (optional)]
Apply to column. Wash 2x with RPE. Elute in 20 ul RNase-free water.
?. [Isolation of Nuclei via Dounce Homogenization and Density Gradient Centrifugation]
If the tissue you are using is difficult to homogenize (for example heart, muscle, etc) then you may want to consider pre-crushing the tissue prior to Dounce homogenization. To do this, pre-chill a mortar and pestle and use the pestle to break the tissue chunk into smaller pieces by repeatedly hitting the tissue. We dont recommend pulverizing the tissue into a powder - it should remain as small chunks.These small chunks may make it so that the Dounce gets stuck more frequently, but in our experience this pre-crushing can help liberate more nuclei from difficult tissues.
?. [Using Nuclei for Single-Nuclei ATAC-seq with 10x Genomics]
Thaw nuclei on ice or use directly after isolation.
?. [Using Nuclei for Single-Nuclei ATAC-seq with 10x Genomics]
Aliquot desired number of nuclei into 1 ml RSB-T (RSB buffer - 10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2 - containing 0.1% Tween). We have had success using 65,000 nuclei but this is far more than recommended by 10x. The number of nuclei you use will affect the doublet rate of your single cell data.
?. [Using Nuclei for Single-Nuclei ATAC-seq with 10x Genomics]
Pellet at 500 RCF at  for  in a fixed-angle rotor. You should be able to see a small pellet if you used more than 25,000 nuclei.
4 °C
?. [Using Nuclei for Single-Nuclei ATAC-seq with 10x Genomics]
Aspirate supernatant using 2 pipetting steps as above (p1000 then p200).
?. [Using Nuclei for Single-Nuclei ATAC-seq with 10x Genomics]
Add 12 ul of 1x Nuclei buffer (The 10x kit provides a 20x stock and you dilute it in water to make 1x buffer).
?. [Using Nuclei for Single-Nuclei ATAC-seq with 10x Genomics]
Transfer 5 ul of nuclei suspension containing nuclei into 10 ul of transposition mix from the 10x protocol.
?. [Using Nuclei for Single-Nuclei ATAC-seq with 10x Genomics]
Mix by pipetting, do not centrifuge or vortex and avoid introducing bubbles. Proceed to transposition incubation as indicated in the part of the 10x protocol.
?. [Using Nuclei for Single-Nuclei ATAC-seq with 10x Genomics]
After this step, you will use 5 ul in the transposition reaction. If you used 65,000 nuclei initially, then 5 ul will contain approximately 27,000 nuclei. Again, this is more than recommended by 10x.