TITLE: Single Nuclei extraction from frozen brain
AUTHORS: Bruno Benitez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;:UNDERLINE;">Protocol: Purification of nuclei from human brains using an iodixanol gradient</span></div><div class = "text-block">Use BSL2 precautions. Samples are not known to be infective, but should be treated as such. Gloves, mask if outside hood, disposable gown. Every surface and reusable equipment (homogenizers, centrifuge tubes) washed with 25% bleach.</div><div class = "text-block">Discarded supernatants or other tissue byproducts should go into 10% bleach for sterilization.</div><div class = "text-block">Discarded tubes and tips go into biohazard waste stream.</div><div class = "text-block"><span style = "font-weight:bold;">Solutions:</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Stock      optiprep 60% Iodixanol </span><span>[Sigma-Aldrich D1556-250mL]</span></li><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Dilutant:</span></li><li style = "counter-reset:ol0;"><span>Final       Concentrations: 900 mM KCL; 30 mM MgCl</span><span style = "vertical-align:sub;">2</span><span>; 120 mM Tricine-KOH       (pH 7.8)</span></li><li style = "counter-reset:ol0;">Recipe       for 50 mls</li><li style = "counter-reset:ol0;">1)  22.5 ml of 2M KCL, +</li><li style = "counter-reset:ol0;">2) 1.5 ml of 1M MgCl2, + </li><li style = "counter-reset:ol0;">3) 6 ml of 1 M Tricine-KOH </li><li style = "counter-reset:ol0;">4) brought up to 50 ml with molecular biology grade water. </li><li style = "counter-reset:ol0;">Components</li><li style = "counter-reset:ol0;">1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).</li><li style = "counter-reset:ol0;">2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion.</li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Homogenization      medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): </span></li><li style = "counter-reset:ol0;">Recipe       (stable at 4C) for 100 mls / 50 mls</li><li style = "counter-reset:ol0;">1) .25 M Sucrose (8.56 g / 4.28g ) </li><li style = "counter-reset:ol0;">2) 150 mM KCL (7.5 ml / 3.75of 2M)</li><li style = "counter-reset:ol0;"><span>3) 5 mM MgCl</span><span style = "vertical-align:sub;">2</span><span> (.5 ml / .25 ml of 1M)</span></li><li style = "counter-reset:ol0;">4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)</li><li style = "counter-reset:ol0;"><span>5) ddH</span><span style = "vertical-align:sub;">2</span><span>O up to 100 ml /50 ml in RNAse free H20</span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">Supplementation (right before       use): </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">1)</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">For 4 samples make 35 mls of homogenization buffer and supplement with:</span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">1.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermine, </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">2.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermidine, </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">3.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">35 ul RNASIN </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">4.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">EDTA free protease inhibitor tablet.</span><span style = "font-weight:bold;"> </span></li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Iodixanol      Dilutions:</span></li><li style = "counter-reset:ol0;">50%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. </li><li style = "counter-reset:ol0;">2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol</li><li style = "counter-reset:ol0;">3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.</li><li style = "counter-reset:ol0;">35%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.</li><li style = "counter-reset:ol0;">2) Scale: need 4 ml per sample</li><li style = "counter-reset:ol0;">3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.</li><li style = "counter-reset:ol0;">Resupsension      Buffer for Nuclei Wash (new from 10x Genomics recipe)</li></ol></div><div class = "text-block">1)  22.5 ml of 2M KCL, +</div><div class = "text-block">2) 1.5 ml of 1M MgCl2, + </div><div class = "text-block">3) 6 ml of 1 M Tricine-KOH </div><div class = "text-block">4) brought up to 50 ml with molecular biology grade water. </div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Components</li><li style = "counter-reset:ol0;">1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).</li><li style = "counter-reset:ol0;">2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion.</li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Homogenization      medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): </span></li><li style = "counter-reset:ol0;">Recipe       (stable at 4C) for 100 mls / 50 mls</li><li style = "counter-reset:ol0;">1) .25 M Sucrose (8.56 g / 4.28g ) </li><li style = "counter-reset:ol0;">2) 150 mM KCL (7.5 ml / 3.75of 2M)</li><li style = "counter-reset:ol0;"><span>3) 5 mM MgCl</span><span style = "vertical-align:sub;">2</span><span> (.5 ml / .25 ml of 1M)</span></li><li style = "counter-reset:ol0;">4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)</li><li style = "counter-reset:ol0;"><span>5) ddH</span><span style = "vertical-align:sub;">2</span><span>O up to 100 ml /50 ml in RNAse free H20</span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">Supplementation (right before       use): </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">1)</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">For 4 samples make 35 mls of homogenization buffer and supplement with:</span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">1.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermine, </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">2.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermidine, </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">3.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">35 ul RNASIN </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">4.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">EDTA free protease inhibitor tablet.</span><span style = "font-weight:bold;"> </span></li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Iodixanol      Dilutions:</span></li><li style = "counter-reset:ol0;">50%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. </li><li style = "counter-reset:ol0;">2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol</li><li style = "counter-reset:ol0;">3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.</li><li style = "counter-reset:ol0;">35%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.</li><li style = "counter-reset:ol0;">2) Scale: need 4 ml per sample</li><li style = "counter-reset:ol0;">3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.</li><li style = "counter-reset:ol0;">Resupsension      Buffer for Nuclei Wash (new from 10x Genomics recipe)</li></ol></div><div class = "text-block">1) Tricine-KOH is 17.92 grams Tricine (Sigma T0377) and 100 mL RNAse free water for 1 M solution (pH to 7.8 with KOH as needed. Usually takes about 3-4 mL of 3M KOH).</div><div class = "text-block">2) 1 M MgCl2 (AM9530G) and 2M KCl (AM9460G) are commercial RNAse free stocks from Ambion.</div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Homogenization      medium (need 6 ml per sample + ~ 2ml extra for 30% Iodixanol): </span></li><li style = "counter-reset:ol0;">Recipe       (stable at 4C) for 100 mls / 50 mls</li><li style = "counter-reset:ol0;">1) .25 M Sucrose (8.56 g / 4.28g ) </li><li style = "counter-reset:ol0;">2) 150 mM KCL (7.5 ml / 3.75of 2M)</li><li style = "counter-reset:ol0;"><span>3) 5 mM MgCl</span><span style = "vertical-align:sub;">2</span><span> (.5 ml / .25 ml of 1M)</span></li><li style = "counter-reset:ol0;">4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)</li><li style = "counter-reset:ol0;"><span>5) ddH</span><span style = "vertical-align:sub;">2</span><span>O up to 100 ml /50 ml in RNAse free H20</span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">Supplementation (right before       use): </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">1)</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">For 4 samples make 35 mls of homogenization buffer and supplement with:</span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">1.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermine, </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">2.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermidine, </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">3.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">35 ul RNASIN </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">4.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">EDTA free protease inhibitor tablet.</span><span style = "font-weight:bold;"> </span></li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Iodixanol      Dilutions:</span></li><li style = "counter-reset:ol0;">50%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. </li><li style = "counter-reset:ol0;">2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol</li><li style = "counter-reset:ol0;">3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.</li><li style = "counter-reset:ol0;">35%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.</li><li style = "counter-reset:ol0;">2) Scale: need 4 ml per sample</li><li style = "counter-reset:ol0;">3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.</li><li style = "counter-reset:ol0;">Resupsension      Buffer for Nuclei Wash (new from 10x Genomics recipe)</li></ol></div><div class = "text-block">1) .25 M Sucrose (8.56 g / 4.28g ) </div><div class = "text-block">2) 150 mM KCL (7.5 ml / 3.75of 2M)</div><div class = "text-block"><span>3) 5 mM MgCl</span><span style = "vertical-align:sub;">2</span><span> (.5 ml / .25 ml of 1M)</span></div><div class = "text-block">4) 20 mM Tricine-KOH (2 ml / 1 ml of 1M)</div><div class = "text-block"><span>5) ddH</span><span style = "vertical-align:sub;">2</span><span>O up to 100 ml /50 ml in RNAse free H20</span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">Supplementation (right before       use): </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">1)</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">For 4 samples make 35 mls of homogenization buffer and supplement with:</span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">1.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermine, </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">2.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermidine, </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">3.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">35 ul RNASIN </span></li><li style = "counter-reset:ol0;"><span style = "font-weight:bold;">4.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">EDTA free protease inhibitor tablet.</span><span style = "font-weight:bold;"> </span></li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Iodixanol      Dilutions:</span></li><li style = "counter-reset:ol0;">50%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. </li><li style = "counter-reset:ol0;">2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol</li><li style = "counter-reset:ol0;">3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.</li><li style = "counter-reset:ol0;">35%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.</li><li style = "counter-reset:ol0;">2) Scale: need 4 ml per sample</li><li style = "counter-reset:ol0;">3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.</li><li style = "counter-reset:ol0;">Resupsension      Buffer for Nuclei Wash (new from 10x Genomics recipe)</li></ol></div><div class = "text-block"><span style = "font-weight:bold;">1)</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">For 4 samples make 35 mls of homogenization buffer and supplement with:</span></div><div class = "text-block"><span style = "font-weight:bold;">1.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermine, </span></div><div class = "text-block"><span style = "font-weight:bold;">2.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">3.5 ul spermidine, </span></div><div class = "text-block"><span style = "font-weight:bold;">3.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">35 ul RNASIN </span></div><div class = "text-block"><span style = "font-weight:bold;">4.</span><span style = "font-weight:bold;font-weight:bold;"> </span><span style = "font-weight:bold;">EDTA free protease inhibitor tablet.</span><span style = "font-weight:bold;"> </span></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"> </li><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Iodixanol      Dilutions:</span></li><li style = "counter-reset:ol0;">50%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. </li><li style = "counter-reset:ol0;">2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol</li><li style = "counter-reset:ol0;">3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.</li><li style = "counter-reset:ol0;">35%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.</li><li style = "counter-reset:ol0;">2) Scale: need 4 ml per sample</li><li style = "counter-reset:ol0;">3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.</li><li style = "counter-reset:ol0;">Resupsension      Buffer for Nuclei Wash (new from 10x Genomics recipe)</li></ol></div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;"><span style = ":UNDERLINE;">Iodixanol      Dilutions:</span></li><li style = "counter-reset:ol0;">50%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. </li><li style = "counter-reset:ol0;">2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol</li><li style = "counter-reset:ol0;">3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.</li><li style = "counter-reset:ol0;">35%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.</li><li style = "counter-reset:ol0;">2) Scale: need 4 ml per sample</li><li style = "counter-reset:ol0;">3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.</li><li style = "counter-reset:ol0;">Resupsension      Buffer for Nuclei Wash (new from 10x Genomics recipe)</li></ol></div><div class = "text-block">1) Recipe: 5 volumes stock (60%) optiprep + 1 volume of dilutant. </div><div class = "text-block">2) Scale: need 2 ml per sample tube + 3 ml per sample for 35% Iodixanol</div><div class = "text-block">3) Example: 4 samples add 17.5 ml stock to 3.5 ml diluant.</div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">35%       Iodixanol </li><li style = "counter-reset:ol0;">1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.</li><li style = "counter-reset:ol0;">2) Scale: need 4 ml per sample</li><li style = "counter-reset:ol0;">3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.</li><li style = "counter-reset:ol0;">Resupsension      Buffer for Nuclei Wash (new from 10x Genomics recipe)</li></ol></div><div class = "text-block">1) Recipe: 7 ml 50% Iodixanol + 3 ml supplemented homogenization medium.</div><div class = "text-block">2) Scale: need 4 ml per sample</div><div class = "text-block">3) For 4 samples add 5.14 mL supplemented homogenization buffer to 12 mL of 50% Iodixanol.</div><div class = "text-block"><ol style = "list-style-type: decimal;"></ol></div><div class = "text-block"> i. 1x PBS with 1.0% BSA and 0.2 U/uL</div><div class = "text-block">1) Recipe:___________ commercial PBS (46-013CM 10x PBS from Corning)</div><div class = "text-block">2) Recipe: __________ BSA (001000162 from Jackson Immunoresearch)</div><div class = "text-block">3) Recipe: ____1:1000______ RNAsin</div><div class = "text-block"><span style = "font-weight:bold;">Protocol: </span></div><div class = "text-block"><span>1. Turn on the Beckman Allegra 64R, and Eppendorf 5430R Centrifuges so they can get to 4</span><span style = "vertical-align:super;">o</span><span style = "vertical-align:super;">.</span></div><div class = "text-block"> i. For Beckman put in swing-out rotor (S0410).</div><div class = "text-block">***Make sure acceleration and deceleration are set to 0 on Beckman*** </div><div class = "text-block">2. Supplement the homogenization medium.</div><div class = "text-block"> i. Add fresh .15mM spermine (1:10,000) (1 ul per 10 ml)</div><div class = "text-block"> ii. Add fresh .5mM spermidine (1:10,000) (1 ul per 10 ml)</div><div class = "text-block"> iii. Add 1 µl RNASIN per ml of homogenization medium if using for RNA</div><div class = "text-block"> iv. Add EDTA free protease inhibitor tablet </div><div class = "text-block">3. Prepare working concentrations of rest of solutions.</div><div class = "text-block">4. Aliquot 2ml of supplemented homogenization medium per 15 ml homogenization tube for each brain. We have used 1g of tissue successfully to yield 15-20 million nuclei. We also used 2g and double that. </div><div class = "text-block">5. Check weight of brain sample and keep on ice in 2ml supplemented homogenization medium.</div><div class = "text-block">6. Homogenize the brain tissue in the 2 ml supplemented homogenization medium.</div><div class = "text-block"> i. Be careful at all steps to treat all samples exactly equally with regard to handling, as different cell type’s nuclei might be more or less vulnerable to physical destruction. All pipetting and resuspension steps should be gentle and consistent to avoid shearing and clumping of nuclei.</div><div class = "text-block"> ii. Put tissue into 15 ml Kontes glass homogenizer tubes</div><div class = "text-block"> iii. Homogenize 10 strokes on ice with ‘A’, and 10 strokes on ice with ‘B’ pestle. </div><div class = "text-block"> i. Use in BSL2 space for human tissue (currently using chem fume hood, with diapers).</div><div class = "text-block"> iv. Check to see that no large pieces have escaped homogenization.</div><div class = "text-block"> v. Transfer pipette with 2ml strippette the homogenate equally into 2 x 2 ml siliconized snap cap microcentrifuge tubes. </div><div class = "text-block"> vi. Add another 500ul supplemented homogenization buffer to the glass homogenizer and transfer remaining homogenate to the same microcentrofuge tubes.</div><div class = "text-block"> i. After use with human tissue, decontaminate homogenizers with 10% bleach during a break in the protocol. Dispose of diapers, tips, etc, in BSL2 waste.</div><div class = "text-block"> ii. Then RNASe Zap, then rinse with ethanol then ddh20 water. </div><div class = "text-block"><span>7. Spin tubes at low speed (500 RCF) in the Eppendorf for 5 minutes at 4</span><span style = "vertical-align:super;">o</span><span style = "vertical-align:super;">.</span></div><div class = "text-block">8. Pour off the supernatant, add 1 ml supplemented homogenization medium, and turn over or flick to resuspend.</div><div class = "text-block"> i. Take 100 ul of supernatant and freeze at -80 if you want cytoplasmic RNA later</div><div class = "text-block">9. Repeat step 8.</div><div class = "text-block">10. While spinning you can add 4ml of 35% Iodixanol to a centrifuge tube for each sample. *Mix the solution up and down to mix again after it has been sitting*</div><div class = "text-block"> i. These are polycarbonate centrifuge tubes from Beckman Coulter, stored in cabinet under the Sorvall in the shared equipment room. </div><div class = "text-block"> ii. May need to do a quick spin of the 35% to get it to settle down if some is stuck on the walls.</div><div class = "text-block">11. Resuspend each pellet in 700 uls of supplemented homogenization buffer. Combine all tubes for each sample into a single 15 ml conical. </div><div class = "text-block">12. Add an equal volume of 50% Iodixanol to the samples in the 15 ml conicals. This will create a final 25% Iodixanol. *Mix 50% Iodixanol right before usage*</div><div class = "text-block">13. Pipette the samples up and down with low retention pipette tips and carefully layer the ~ 3ml-4mls of 25% Iodixanol (2 tubes) on top of the 4 mL of 35% Iodixanol using a low attachment tip (silver box). You will need to use the same volume of all samples so that tubes are balanced, so scale to your smallest sample.</div><div class = "text-block"> i. Layering is best done by holding the bottle almost horizontal and using a P1000 to pipette slowly into the wall of the tube slightly above the 35 % Iodixanol. It is important to make sure the layers do not mix. </div><div class = "text-block">14. Centrifuge the tubes at 10,000 rpm (top speed) for 30 minutes in the Beckman Allegra *Check acceleration and deceleration again*</div><div class = "text-block">15. Carefully remove the centrifuge tubes. Take back to BSL2 space. The nuclei should be visible as a cloudy layer in the interface of the 25%/35% Iodixanol. with a transfer pippette, remove the top pink debris layer of myelin (suck off with a transfer pipette).</div><div class = "text-block">16. With a low retention pipette remove the nuclei by vacuuming them up from just above the layer. Then place into a 1.5mL Eppendorf – NONSTICK TUBES (for nuclei). No more than 500 uls per tube. Usually 2 tubes per sample. </div><div class = "text-block">17. Add 1 ml of supplemented Nuclei Wash buffer to the tubes & mix. </div><div class = "text-block">18. Centrifuge at 500g’s (rcf) at 4o.</div><div class = "text-block">19. Nuclei should be pelleted at the bottom</div><div class = "text-block">20. Remove & discard supernatant </div><div class = "text-block">21. Resuspend nuclei in about 750 mls of Nuclei Wash buffer. Bring together all nuclei from the same sample into a single tube.</div><div class = "text-block">22. Centrifuge at 500g’s (rcf) at 4o.</div><div class = "text-block">23. Nuclei should be pelleted at the bottom</div><div class = "text-block">24. Remove & discard supernatant </div><div class = "text-block">25. Resuspend nuclei in about 1000 mls of Nuclei Wash buffer</div><div class = "text-block">26. Remove & discard supernatant </div><div class = "text-block">27. Resuspend nuclei in about 1000 ul of Nuclei Wash buffer. </div><div class = "text-block">28. Let it drip through a 40 um Nylon mesh cell strainer (#229481)to remove clumps per 10x protocol.</div><div class = "text-block">29. Take out 20 uls, mix 1:1 with a 1:2000 DAPI solution, and count on hemacytometer or countess and confirm lack of clumps.</div><div class = "text-block">30. Adjust aliquot of nuclei to desired dilution (1,200 cells per ul). Count again to confirm dilution.</div><div class = "text-block">31. The rest may be stored as follows: Pellet and resuspend in 100% methanol</div></div>

[STEPS]