FixNCut v1.0
Luciano G Martelotto, Laura Jiménez-Gracia, Domenica Marchese, Holger Heyn
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Abstract
This protocol details reversible fixation for cells and tissues for subsequent use in sc/snRNA, sc/snATAC or Multiome. Spatial-Omics compatibility is being validated. For more information check this preprint: https://www.biorxiv.org/content/10.1101/2023.06.16.545221v2
Attachments
Steps
Preparation of DSP (Oz Soup) stock and working solutions
Room temperature for 0h 30m 0s and then prepare 50x stock solution of DSP (50mg/mL) in molecular biology grade dimethyl sulfoxide (Sigma, cat. no. D8418-50ML). Dispense the stock into 100µL aliquots and store at -80°C.
Immediately before use prepare 500µL of 1mg/mL DSP working solution (DSP 1x is also known as OzSoup) in molecular biology grade 1x PBS as follows: aliquot 10µL of stock DSP in a 1.5mL eppi tube and while vortexing (VERY IMPORTANT) add 490µL of PBS (Room temperature) dropwise using a P200.
Filter DSP working solution using a 40-μm Flowmi strainer (Sigma, cat. no. BAH136800040-50EA).
Fixation
Submerge a ~3x3 mm (the smaller the better) piece of tissue (or organoids) in 500µL of the OzSoup and incubate for 0h 45m 0s at Room temperature.
For cells in suspensions, wash cells in cold PBS at least twice (no media + FBS should be present). Pellet cells and resuspend (up to 2 milliion) cells in 500µL of the Oz soup and incubate for 0h 30m 0s at Room temperature.
At the 45 min mark, add 10µL of 1Molarity (M) Tris-HCl 7.5, mix well by vortexing for 2-3” and sit at Room temperature for at least 0h 15m 0s.
Pellet the pieces of tissue at 500rcf or a 5-10” in minispinner, and remove supernatant.
For cells, mix by vortexing 2-3min and pellet cells 500rcf .
Add 1mL of PBS, mix by vortexing 2-3 min, and pellet pieces (or cells) as above, remove supernatant.
For cells, mix by vortexing 2-3 min and pellet cells 500rcf.
Fixation: For cells only
Repeat 8 once more for a total of 2 washes. Continue on step 18-21 below. If sorting or shipping samples follow step 17.
Fixation: For tissues only
Add 1mL of 200µg/mL Liberase (Liberase™ Research Grade Sigma-Aldrich 5401127001, https://www.sigmaaldrich.com/US/en/product/roche/libtmro) in PBS (80µL of 2.5mg/mL Liberase + 920µL PBS).
Incubate at 37°C for 0h 30m 0s with agitation 800rpm. At 15 min mark pipette up and down 5 times.
After digestion, filter the digestion reaction through a 70 µm mesh.
Add 10mL * ice-cold PBS and pellet cells for 500rcf,4°C (swinging bucket rotor). Pre-Wash.
Remove supernatant and add 10mL* of cold PBS+1% BSA and resuspend the pellet before pelleting again (500rcf,4°C). Wash 1.
Remove supernatant and add 10mL* of cold PBS+1% BSA and resuspend the pellet before pelleting again (500rcf,4°C). Wash 2.
Remove supernatant and add 10mL* of cold PBS+1% BSA and resuspend the pellet before pelleting again (500rcf,4°C). Wash 3.
Optional : If storing for later processing or shipping samples, freeze cells using a cryopreservation strategy as if the main goal was to keep the cells alive. For example, use CryoStor10 and Mr Frosty for slow freezing. Include 1-2 U/uL of RNAse inhibitor per sample for storage. Store -80°C until use. After storage, thaw in water bath at 37°C and wash cells twice with PBS+0.5-1%BSA.
Remove supernatant and resuspend cells in 0.5-1mL of PBS+1% BSA (optionally add +0.5-1 U/uL RNAse Inhibitor).
Filter cells through Flowmi 40 um.
Count cells and bring concentration to 1000-1500 cells/uL.
Load Chromium as per manual.
