TITLE: Nuclei Isolation from Tissue for 10x snRNA
AUTHORS: Minyi Shi, Annika Weimer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Nuclei Isolation from Tissue for 10x snRNA</div></div>

[STEPS]
?. [Stock Buffers]
?. [Before you start the protocol:]
1)All steps should be performed on ice or at 4°C. Pre-chill a swinging bucket centrifuge and a fixed angle centrifuge to 4°C.2)Pre-chill all Dounces and pestles to 4°C in a fridge.3)Pre-chill all tubes. For each sample you are processing, you will need:a.One 2 ml round-bottom LoBind tube for gradient separationb.One 50 ml conical for filtration step (often optional)4)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 and buffers are stored at 4°C.a.Remember that the catalog number provided for iodixanol from Sigma comes as a 60% solution (not 100%).5)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:]
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 2 ml Dounce containing 1 ml cold 1x HB (add 15 ul RiboLock per ml of1x HB and mix well).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 (~20 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 (Fisher Scientific Cat: 22363547) and transfer homogenate to a pre-chilled 50 ml tube. Transfer homogenate to 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 RCF 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 pipetting14)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 excessIodixanol 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 RCF with the brake off. Handle tubes gently so as to not disturb the gradient.a.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.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 (1X PBS with 1.0% BSA and 0.2U/µl RNase Inhibitor).Mix gently by pipetting. Filter nuclei suspension to 1.5 ml LoBind tube with 20uM Mini-Strainer (PluriSelect Cat: 431002040).20)Centrifuge at 500 rcf for 5 min at 4°C.21)Remove supernatant, resuspend nuclei in 100 ul of wash buffer.22)Determine the nuclei concentration using a Countess Automated Cell Counter or manualcounting using microscope.23)Dilute nuclei to 800-1000 nuclei /ul using nuclease free water.24)Proceed immediately to Chromium Next GEM Single Cell 3’ Reagent Kits User Guide(CG000204)