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==Protocol== #Received nuclei in 15 mL eppendorf tubes containing ~1 mL of PBSE/BSA ##PBSE-PBS+ 2mM EGTA; BSA 1% #Centrifuge in bucket rotor at 900xg for 10 min at 4C #Remove supernatant and resuspend nuclei in 100 uL PBSE/BSA + 10% DMSO; pipette gently, do not use P10 or P2.5 ##Optional step for filtration with 20 um filter; skipped this step #Count Nuclei with cell counter #Aliquot and freeeze down to -80C in styrofoam box or Mr. Frosty ##Store at -80C, use within a few months ##When thawing, thaw rapidly in hand, gently pipette 6-8 times using P20 and add reagent quickly ===Nuclei Counting and Aliquots=== {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> |- style="background-color:#B7DEE8;font-size:12pt" align="center" | width="65" height="30" | Species | width="81" | Nuclei Count (#/mL) | width="65" | Volume (uL) | width="65" | Total Nuclei Received | width="82" | Volume of Aliquots (uL) | width="65" | Number of Aliquots | width="77" | Nuclei/Aliquot |- style="font-size:12pt" valign="bottom" | height="15" | Mouse | align="center" | 5.36E+06 | align="center" | 100 | align="center" | 5.36E+05 | align="center" | 10 | align="center" | 10 | align="center" | 5.36E+04 |- style="background-color:#D9D9D9;font-size:12pt" valign="bottom" | height="15" | Human | align="center" | 1.75E+07 | align="center" | 500 | align="center" | 8.75E+06 | align="center" | 30 | align="center" | 15 | align="center" | 5.25E+05 |}
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