TITLE: Protocol to isolate and fix nuclei from flash frozen mouse gastrocnemius for IGVF
AUTHORS: Elisabeth Rebboah

[DESCRIPTION]
This protocol describes isolation of nuclei from 10 week old left or right mouse gastrocnemius muscle (tissue ID: 16) from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using the Parse Biosciences protocol (Split-seq). We process 1 rep from each strain per day; e.g. female rep 1 across all 8 strains. For 8 samples, this protocol takes about 3.5 hours from start to finish.

The results is 1 aliquot of a fixed single-nucleus suspension for Parse Bio snRNA-seq ("Split-seq") from each of the 8 samples at >= 2,500 nuclei/ul stored at -80C.

The first part of the protocol describes tissue lysis and nuclei extraction using Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. It also includes debris removal using Miltenyi Biotec’s Debris Removal Solution and extra filtering steps specifically for working with skeletal muscle tissue. The second part describes nuclei fixation using Parse Biosciences Evercode Nuclei Fixation Kit with v2 reagents. Due to low nuclei recovery, we modify the original Parse Biosciences Evercode fixation protocol (attached) by using half volumes of all fixation reagents. We do not fix extra nuclei for other assays such as SHARE-seq, but save the whole left or right muscle. 

[GUIDELINES]
Tilt tube and slowly add PBS during debris removal. Ideally, the cloudy debris is only in the band rather than the nuclei layer.
We recommend using a 5 mL pipette for aspirations and resuspensions > 1 mL.
Record everything in the IGVF spreadsheet, “Samples into experiment” tab.

[STEPS]
SECTION: Setup
2. Pre-chill centrifuge to 4C.
SECTION: Setup
3. Prepare 2 large ice buckets.
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4. Prepare 35 ml lysis buffer on ice in a 50 mL conical tube. Distribute 2 mL into 8 gentleMACS C Tubes on ice. Add 175 ul RNase inhibitor to the lysis buffer aliquot the day of the experiment.
SECTION: Setup
5. Prepare 3.5 ml NB + BSA. Add 44.1 ul RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment.
SECTION: Setup
6. Prepare 50 mL RSB on ice in 2 50 mL conical tubes. We keep a larger amount of PBS + 0.1% BSA at 4C, adding the RNase inhibitor the day of the experiment.
SECTION: Setup
7. Prepare 5 ml nuclei buffer + RNase inhibitor for final resuspension. Add 44.1 ul RNase inhibitor to 5 ml nuclei buffer.
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8. Take an aliquot of PBS out of 4C and keep on ice.
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9. Take an aliquot of Debris Removal Solution out of 4C and keep on ice.
SECTION: Setup
10. Thaw components of 1 Parse Biosciences Nuclei Fixation kit at room temperature, then place on ice.
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11. Distribute 10 ul NucBlue Fixed Cell ReadyProbes into 24 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, 8 tubes for counting after fixation, and another 8 tubes for filtered fixed nuclei.
SECTION: Tissue lysis and nuclei extraction
12. Keep flash frozen tissue samples on dry ice.
SECTION: Tissue lysis and nuclei extraction
13. Prepare 6 well plates on ice with ~2 ml of HBSS per well.
SECTION: Tissue lysis and nuclei extraction
14. If necessary, drop both gastrocnemius tissues in a well, let them melt slightly, and separate them carefully using forceps.
SECTION: Tissue lysis and nuclei extraction
15. Return one muscle to the sample tube and move the other to another labeled 1.5 ml tube. Flash-freeze both in liquid nitrogen.
SECTION: Tissue lysis and nuclei extraction
16. Proceed with only one. Keep the other frozen in the same sample tube and return tubes to -80C box.
SECTION: Tissue lysis and nuclei extraction
17. Drop left or right frozen tissue into a chilled gentleMACS C Tube with 2 mL lysis buffer. Close tubes firmly and invert immediately, ensuring tissue is not stuck to the bottom or side. Keep tubes on ice and proceed immediately to dissociation.
SECTION: Tissue lysis and nuclei extraction
18. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes).
SECTION: Tissue lysis and nuclei extraction
19. Remove tubes, ensuring tissue did not get stuck on the sides, and spin down in a 4C centrifuge for ~10 seconds to bring liquid to the bottom, then place tubes back on ice.
SECTION: Tissue lysis and nuclei extraction
20. Filter nuclei suspension through 70 um MACS SmartStrainer into a 5 mL tube. Fit a tube rack in ice for extra stability while filtering.
SECTION: Tissue lysis and nuclei extraction
21. Wash 70 um MACS SmartStrainer with 2 mL additional lysis buffer. Add 2 mL to C tubes, cap, and swish to recover any nuclei stuck to the sides and cap of the C tubes, then wash the strainer.
SECTION: Tissue lysis and nuclei extraction
22. Discard strainer and centrifuge the nuclei suspension at 4C, 350g for 5 minutes. 
SECTION: Tissue lysis and nuclei extraction
23. Aspirate supernatant and resuspend nuclei pellet in 3.1 mL RSB.
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24. Filter nuclei suspension through 30 um MACS SmartStrainer into a 15 mL tube.
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25. Add 900 ul Debris Removal Solution and mix by pipetting 10 times slowly up and down using a 5 mL pipette.
SECTION: Tissue lysis and nuclei extraction
26. Overlay with 4 ml PBS using a P1000 or 5 mL pipette (whichever you are more comfortable with).Tilt tube 45 degrees and slowly add the first mL. You can increase speed after the first mL of PBS is added.
SECTION: Tissue lysis and nuclei extraction
27. Centrifuge at 4C, 3000g for 10 minutes with full acceleration and no brake. Three phases are formed: top clear buffer layer, cloudy debris band, and clear layer containing nuclei. Pellet usually visible.
SECTION: Tissue lysis and nuclei extraction
28. Aspirate the two top phases (buffer layer and all cloudy debris band) and discard. Aspirate the first phase, then the second phase. Stay above the third layer of nuclei to prevent loss. (See Fig. 1.)

  

SECTION: Tissue lysis and nuclei extraction
29. Fill with cold RSB to a final volume of 5 mL.
SECTION: Tissue lysis and nuclei extraction
30. Gently invert the tube three times. Do not vortex.
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31. Centrifuge at 4C, 1000g for 10 minutes with full acceleration and full brake.
SECTION: Tissue lysis and nuclei extraction
32. Aspirate supernatant completely.
SECTION: Tissue lysis and nuclei extraction
33. Resuspend cells carefully in 375 ul NB-BSA + RNase inhibitor and filter through a 40 um strainer into a new 5 mL tube.
SECTION: Tissue lysis and nuclei extraction
34. Count nuclei. Use 1:2 dilution factor, 10 ul + 10 ul dye.
SECTION: Nuclei fixation
35. Add 125 uL Nuclei Fixation Solution to the filtered nuclei in 375 ul and mix 3 times. Do not over-mix.
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36. Incubate nuclei for 10 minutes on ice. Set 2 P200 pipettes to 40 ul and 125 ul.
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37. Add 40 uL Nuclei Permeabilization Solution and mix by pipetting 3 times with the P200 still set to 125 uL. Do not over-mix.
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38. Incubate 3 minutes with nuclei on ice.
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39. Add 2 mL Nuclei Neutralization Solution and invert the tube once to mix. 
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40. Centrifuge at 4C, 750g for 10 minutes.
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41. Aspirate and discard supernatant.
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42. Resuspend the samples in 500 uL Nuclei Buffer with RNase inhibitor without BSA. Check concentration with a hemocytometer under the microscope. Use 1:2 dilution factor, 10 ul + 10 ul dye.
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43. Filter nuclei through a 20 um filter in 1, 2, 3, or 4 rounds depending on the amount of debris. Place filter in labeled 1.5 ml tube and dispense nuclei in 500 ul on top. Centrifuge at 4C, 200g for 1 minute to pull the solution through the filter. Repeat step if necessary, using a new filter for each round. Our reasoning is to prevent clogging by filtering in multiple rounds, but yield decreases by 90% before and after fixation, mostly due to the filtration at this step.
SECTION: Nuclei fixation
44. Take a 10 ul aliquot to dilute 1:2 with prepared 10 ul dye to manually count with a disposable hemacytometer and record numbers.
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45. Count nuclei. Use 1:2 dilution factor, 10 ul + 10 ul dye.
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46. Re-concentrate: spin nuclei 750g for 5 minutes and carefully take off supernatant until 50 ul are remaining. Resuspend (hopefully visible) pellet in the remaining 50 ul.
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47. Add Nuclei DMSO: 1 ul into 50 ul samples and gently flick tubes to mix. One minute later, add another 1 ul and flick to mix, then after another minute add a final 1 ul for a total volume of 3 ul. Mix by gently pipetting 5x with a P200 set to 25 ul.
SECTION: Nuclei fixation
48. Place tubes in a Mr. Frosty for storage at -80C. The next day, move tubes to boxes in -80C racks.
SECTION: Setup
1. Label tubes.