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 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. We process 1 rep from each strain per day; e.g. female rep 1 across all 8 strains. The main products we use are Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish.

The results are 1 aliquot of fixed single-nucleus suspensions for Parse per each of the 8 samples at >= 2,500 nuclei/ul.


[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
1. Pre-chill centrifuge to 4C.
SECTION: Setup
2. Prepare 2 large ice buckets.
SECTION: Setup
3. Prepare lysis buffer on ice in a 50 mL conical tube. Distribute 2 mL into 8 gentleMACS C Tubes on ice.
SECTION: Setup
4. Prepare NB + BSA + RNAse inhibitor in a 5 mL tube.
SECTION: Setup
5. Prepare RSB on ice in a 50 mL conical tube. We keep a larger amount of PBS + 0.1% BSA at 4C, adding the RNase inhibitor the day of the experiment.
SECTION: Setup
6. Prepare NB + RNase inhibitor in a 5 mL tube for final resuspension.
SECTION: Setup
7. Take an aliquot of PBS out of 4C and keep on ice.
SECTION: Setup
8. Take an aliquot of Debris Removal Solution out of 4C and keep on ice.
SECTION: Setup
9. Thaw components of 1 Parse Biosciences Nuclei Fixation kit at room temperature, then place on ice.
SECTION: Setup
10. 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
11. Keep flash frozen tissue samples on dry ice.
SECTION: Tissue lysis and nuclei extraction
12. Prepare 6 well plates on ice with ~2 ml of HBSS per well.
SECTION: Tissue lysis and nuclei extraction
13. 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
14. 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
15. 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
16. 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
17. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes).
SECTION: Tissue lysis and nuclei extraction
18. 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
19. 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
20. 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
21. Discard strainer and centrifuge the nuclei suspension at 4C, 350g for 5 minutes. 
SECTION: Tissue lysis and nuclei extraction
22. Aspirate supernatant and resuspend nuclei pellet in 3.1 mL RSB.
SECTION: Tissue lysis and nuclei extraction
23. Filter nuclei suspension through 30 um MACS SmartStrainer into a 15 mL tube.
SECTION: Tissue lysis and nuclei extraction
24. 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
25. 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
26. 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
27. 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
28. Fill with cold RSB to a final volume of 5 mL.
SECTION: Tissue lysis and nuclei extraction
29. Gently invert the tube three times. Do not vortex.
SECTION: Tissue lysis and nuclei extraction
30. Centrifuge at 4C, 1000g for 10 minutes with full acceleration and full brake.
SECTION: Tissue lysis and nuclei extraction
31. Aspirate supernatant completely.
SECTION: Tissue lysis and nuclei extraction
32. 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
33. Count nuclei. Use 1:2 dilution factor, 10 ul + 10 ul dye.
SECTION: Nuclei fixation
34. Add 125 uL Nuclei Fixation Solution to the filtered nuclei in 375 ul and mix 3 times. Do not over-mix.
SECTION: Nuclei fixation
35. Incubate nuclei for 10 minutes on ice. Set 2 P200 pipettes to 40 ul and 125 ul.
SECTION: Nuclei fixation
36. Add 40 uL Nuclei Permeabilization Solution and mix by pipetting 3 times with the P200 still set to 125 uL. Do not over-mix.
SECTION: Nuclei fixation
37. Incubate 3 minutes with nuclei on ice.
SECTION: Nuclei fixation
38. Add 2 mL Nuclei Neutralization Solution and invert the tube once to mix. 
SECTION: Nuclei fixation
39. Centrifuge at 4C, 750g for 10 minutes.
SECTION: Nuclei fixation
40. Aspirate and discard supernatant.
SECTION: Nuclei fixation
41. Resuspend the samples in 500 uL Nuclei Buffer without BSA, with RNase inhibitor. Check concentration with a hemocytometer under the microscope. Use 1:2 dilution factor, 10 ul + 10 ul dye.
SECTION: Nuclei fixation
42. 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
43. Take a 10 ul aliquot to dilute 1:2 with prepared 10 ul dye to manually count with a disposable hemacytometer and record numbers.
SECTION: Nuclei fixation
44. Count nuclei. Use 1:2 dilution factor, 10 ul + 10 ul dye.
SECTION: Nuclei fixation
45. 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.
SECTION: Nuclei fixation
46. 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.
SECTION: Nuclei fixation
47. Place tubes in a Mr. Frosty for storage at -80C. The next day, move tubes to boxes in -80C racks.