TITLE: Tissue Nuclei Isolation and Glutaraldehyde Fixation for sci RNA-Seq
AUTHORS: David Fraser Read, Cole Trapnell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details a method for isolating and fixing nuclei from primary tissue such that the resulting fixed nuclei are compatible with sci RNA-Seq. </div><div class = "text-block"><span>A protocol.io page at (</span><a href="https://www.protocols.io/view/sci-rna-seq3-9yih7ue" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/sci-rna-seq3-9yih7ue</span></a><span>) goes over the sci RNA-Seq workflow. However, the method for nuclei preparation in that protocol does not yield usable nuclei when applied to many primary tissues. The protocol here would replace previous nuclei preparation workflows for some tissues.</span></div></div>

[STEPS]
?. [Buffer Preparaion]
Nuclei Buffer: 10 mM Tris HCl, 10 mM NaCl, 3 mM MgCl2 in nuclease-free waterStore at 4C.
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For each sample to dissociate, add 5 mL of lysis/fixation buffer (out of 10 mL available per sample) to a Gentle MACS M Tube. Store both the M Tube and a second tube (holding the remaining 5 mL of buffer) on ice.
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Add frozen tissue (~100-250 mg in total, stored on dry ice up until the point) to the MACS M Tube (which already has 5 mL buffer within).Note: We have used either powdered tissue or small chunks, on the order of
?. [Buffer Preparaion]
Lysis/Fixation Buffer:For each sample to be dissociated, prepare 10 mL of lysis/fixation buffer. Chill on ice.2.5 mL of 4x Hypotonic phosphate buffer500 uL Vanadyl Ribonucleoside Complex (200 mM)20 uL of Triton X-100 (10% in water)20 uL PVSA (.3% solution)2 mL glutaraldehyde (25% solution in water)4.9 mL nuclease-free water100 uL diethyl pyrocarbonate (DEPC) Important: Add immediately before useNote: Store ~500 uL aliquots of vanadyl solution frozen at -20C. When thawing for use, thaw completely in 37-42C water bath. If thawed at room temperature, solution has a tendency to stay precipitated upon thaw.Note: Add DEPC last, and immediately before proceeding to tissue dissociation. DEPC has a short half life and will degrade rapidly if mixed and left sitting. Also, DEPC has a tendency to form an organic layer at the bottom of tubes. Vortex or pipette vigorously to mix into solution, and visually confirm no separation before proceeding.
?. [Buffer Preparaion]
4x Hypotonic Phosphate Buffer: 40 mM NaCl, 12 mM MgCl2, 40 mM sodium phosphate (pH 7.).Store at 4C for up to a month. Note: Solution precipitates over time, so it is best to prepare relatively small amounts and re-make stocks every couple weeks. Check for white precipitate before use.
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10% Triton X-100 StockCombine 1.0 mL of Triton X-100 with 9.0 mL nuclease-free water. Gently rock overnight to mix, store at 4C.
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.3% Polyvinyl Sulfonic Acid (PVSA):Combine 100 uL PVSA (30% stock solution) with 9.9 mL nuclease-free water
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Nuclei suspension buffer (NSB)For each sample to be dissociated, prepare 1.2 mL. Chill on ice1.2 mL Nuclei Buffer (10 mM Tris HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2)12 uL Bovine Serum Albumin (BSA)12 uL SuperaseInNote: 1.2 mL per sample assumes that a single aliquot of fixed nuclei will be frozen and stored per sample. Add 100 uL of volume to this amount for every additional aliquot that you plan to store. (E.g. if planning to split a sample into 3 aliquots to store, make 1.4 mL of buffer for that sample in this step).
?. [Dissociation and filtering]
Quickly cap and seal the M Tube holding tissue + buffer. Place into the Gentle MACS, and run the "m_heart_01.01" protocol (a pre-programmed method).Remove the tube from it's holder, invert once, then re-place the tube and run the cycle again. (In total, the cycle will be run twice).
?. [Dissociation and filtering]
Place dissociated sample in the MACS tube back on ice. In a chemical safety hood, filter the dissociated tissue solution through a 70 uM strainer. Next, use a new pipette tip to wash the strainer with the 5 mL prepared and stored previously (that was not added to the MACS tube).
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Incubate on ice for the remainder of the 15 minute period (time begins when the first cycle of dissociation protocol was started). Invert tubes of filtered nuclei every 5 minutes.
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Start timing here for nuclei fixation. The nuclei should be fixed for 15 minutes, but start timing this period when the first dissociation cycle on the Gentle MACS begins. Note: This is because the filtering in the next step can take longer/shorter depending on number of samples, etc. Do not vary the fixation time as a function of variation in filtering time spent, only fix with respect to (time of dissociation starting + 15 minutes).
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Centrifuge nuclei. Spin 500 RCF, 8 minutes, at 4C. Optional: Consider increasing spin time up to 12 minutes for small samples/if particularly high nuclei recovery numbers are key.
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Remove and dispose of supernatant. Resuspend the pellet gently but thoroughly with 1 mL of NSB (pre-chilled on ice).
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Centrifuge nuclei. Spin 500 RCF, 5 minutes, at 4C.
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Remove and dispose of supernatant. Next, resuspend the nuclei gently but thoroughly in the appropriate volume to make up 100 uL aliquots. Resuspend in (10 + (numer-of-aliquots * 100)) uL. So 110 uL if saving 1 aliquot, 210 uL if saving 2 aliquots, 310 if saving 3 aliquots, etc.Optional: Count nuclei here to guarantee an appropriate number of nuclei are available for later steps. Having
?. [Dissociation and filtering]
Disperse 100 uL to separate tubes to snap-freeze as separate aliquots. Snap freeze in liquid nitrogen, store at -80C or colder.