TITLE: Protocol to isolate and fix nuclei from cryopreserved left cortex mouse bridge samples for IGVF
AUTHORS: Elisabeth Rebboah

[DESCRIPTION]
This protocol describes isolation of nuclei from 10 week old mouse left cerebral cortex from B6J and CASTJ, preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using Parse Biosciences. We process 2 reps from each strain per day; e.g. both male and female reps across both 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 main difference from our other nuclei prep protocols is storage of the tissue: for these mouse bridge samples, tissues were stored in 1 mL Bambanker media in cryotubes.

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

The first part of the protocol describes tissue lysis and nuclei extraction using Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. The second part describes nuclei fixation using Parse Biosciences Evercode Nuclei Fixation Kit with v2 reagents.

[GUIDELINES]
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. Label tubes.
SECTION: Setup
2. Pre-chill centrifuge to 4C.
SECTION: Setup
3. Prepare ice buckets.
SECTION: Setup
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
6. 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
7. Prepare 1.5 ml nuclei buffer + RNase inhibitor for final resuspension. Add 18.6 ul RNase inhibitor to 1.5 ml nuclei buffer.
SECTION: Setup
8. Thaw components of 2 Parse Biosciences Nuclei Fixation v2 kits at room temperature, then place on ice.
SECTION: Setup
9. Distribute 20 ul NucBlue Fixed Cell ReadyProbes into 16 PCR strip tubes for cell counting. Need 8 tubes for counting after nuclei extraction, and another 8 tubes for final fixed nuclei.
SECTION: Tissue lysis and nuclei extraction
10. Thaw tissues in Bambanker media on ice until the tissue can be extracted.
SECTION: Tissue lysis and nuclei extraction
11. Transfer the tissue to a chilled gentleMACS C Tube with 2 ml lysis buffer using forceps. 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
12. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes).
SECTION: Tissue lysis and nuclei extraction
13. 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
14. 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
15. 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
16. Discard strainer and centrifuge the 4 ml nuclei suspension at 4C, 350g for 5 minutes. 
SECTION: Tissue lysis and nuclei extraction
19. Count nuclei. Use 1:11 dilution factor, 2 ul + 20 ul dye.
SECTION: Parse Nuclei Fixation
21. Remove supernatant and and resuspend nuclei in 750 ul NB-BSA + RNase inhibitor and filter through a 40 um strainer (provided in Parse Biosciences kit) into a new 5 ml tube.
SECTION: Parse Nuclei Fixation
23. Incubate nuclei for 10 minutes on ice. Set 1 P200 pipette to 80 ul and keep the P1000 at 250 ul.
SECTION: Parse Nuclei Fixation
24. Add 80 uL Nuclei Permeabilization Solution and mix by pipetting 3 times with the P1000 still set to 250 uL. Do not over-mix.
SECTION: Parse Nuclei Fixation
25. Incubate 3 minutes with nuclei on ice.
SECTION: Parse Nuclei Fixation
26. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. 
SECTION: Parse Nuclei Fixation
27. Centrifuge at 4C, 750g for 10 minutes.
SECTION: Parse Nuclei Fixation
28. Aspirate and discard supernatant.
SECTION: Parse Nuclei Fixation
29. Resuspend the samples in 300 ul Nuclei Buffer with RNase inhibitor without BSA and move through a 40 um filter into a labeled 1.5 ml tube.
SECTION: Parse Nuclei Fixation
30. Count nuclei. Use a 1:11 dilution factor, e.g. 2 ul + 20 ul dye.
SECTION: Parse Nuclei Fixation
31. Add Nuclei DMSO: 5 uL and gently flick tubes to mix. One minute later, add another 5 uL and flick to mix, then after another minute add a final 5 uL for a total volume of 15 uL. Mix by gently pipetting 5x with a P200 set to 150 ul.
SECTION: Parse Nuclei Fixation
32. Split nuclei suspension into 2 labeled tubes, 150 ul per tube.
SECTION: Parse Nuclei Fixation
33. Place tubes in a Mr. Frosty for storage at -80C. The next day, move tubes to boxes in -80C racks.
SECTION: Setup
5. Prepare 25 ml RSB in a 50 ml conical tube on ice. Add 125 ul RNase inhibitor the day of the experiment.
SECTION: Tissue lysis and nuclei extraction
17. Discard supernatant and resuspend nuclei pellet in 3 ml RSB.
SECTION: Tissue lysis and nuclei extraction
18. Filter nuclei suspension through 30 um MACS SmartStrainer into a 5 ml tube.
SECTION: Parse Nuclei Fixation
20. Set aside 4 million nuclei in RSB in a new 5 ml tube and spin down at 4C, 350g for 5 minutes.
SECTION: Parse Nuclei Fixation
22. Add 250 uL Nuclei Fixation Solution and mix 3 times. Do not over-mix.
SECTION: Storage of leftover nuclei
34. Move remaining nuclei in RSB on ice to labeled 2 ml tubes.
SECTION: Storage of leftover nuclei
35. Spin 750g, 4C, 5 minutes.
SECTION: Storage of leftover nuclei
36. Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80C.