1) Remove samples from liquid nitrogen storage and keep on dry ice until use.
2) If you are working with a tissue type that is particularly hard to dissociate via Douncing, it can be helpful to pre-crush (do not pulverize) your tissue fragment using mortar and pestle. The tissue must remain frozen and cold during this entire process. Otherwise proceed to Step 3.
3) Place 20-50 mg frozen tissue or crushed tissue into a pre-chilled 7 ml Dounce containing 1 ml cold 1x HB.
4) Dounce with “A” loose pestle until resistance goes away (~10 strokes).
5) Place “A” pestle into beaker with sterile water to soak for cleaning later.
a. Optional – If residual un-homogenized tissue makes it difficult to Dounce, filter homogenate through a pre-chilled 50 ml conical using a 70 um bucket-style cell strainer filter prior to using tight pestle “B”.
6) Dounce with “B” tight pestle until resistance goes away (~15 strokes).
7) Place “B” pestle into beaker with sterile water to soak for cleaning later.
8) Filter during transfer using a 40-um cell strainer (PluriSelect Cat: 43-10040-60) to a pre-chilled 2 ml LoBind tube.
9) Place Dounce into beaker with sterile water to soak for cleaning later.
10) Pellet nuclei by spinning 5 min at 4°C at 350 xg in a fixed angle centrifuge.
11) Remove all supernatant, if the pellet is not clearly visible, you can leave 50 ul supernatant in the tube.
12) Gently resuspend nuclei in 350 ul 1x HB, but make sure the total volume of nuclei suspension is 400 ul. Make sure nuclei are fully resuspended without clumps.
13) Add 1 volume (400 ul) of 50% Iodixanol Solution and mix well by pipetting
14) 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.
15) 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.
a. 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.
16) In a pre-chilled swinging bucket centrifuge, spin for 20 min at 4°C at 3,000 xg with the brake off. Handle tubes gently so as to not disturb the gradient. Set acceleration level at 1, deceleration level at 0. Set centrifuge time to 25 min and after it’s done, it takes another 13 min to stop.
a. Iodixanol is meant to be used at higher speeds (10,000 xg) but high-speed swinging bucket centrifuges are not always readily available, so we perform this step at 3,000 xg and have not had any issues.
17) 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.
18) 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.
19) Dilute nuclei by adding 200 ul of wash buffer. Mix gently by pipetting. Filter nuclei suspension to 1.5 ml LoBind tube with 40 um Mini-Strainer (PluriSelect Cat: 43-10040-60).
Take a sample and count nuclei using Trypan Blue.
Centrifuge at 500 xg for 5 min at 4°C.
Remove the supernatant without disrupting the nuclei pellet.
Resuspend in x uL chilled Diluted Nuclei Buffer. (The volume can be changed based on counting results from step 13, target for 8,000 nuclei/uL)
Take a sample and count nuclei using Trypan Blue.
Dilute to 3220 nuclei/uL and proceed immediately to 10x single cell Multiome user guide. Use 5 uL nuclei.