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

[DESCRIPTION]
This protocol describes isolation of nuclei from left and right 10 week old mouse ovary and oviduct (pooled tissue ID: 26) 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 Parse Biosciences. 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 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. 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.

[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
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 1.5 ml nuclei buffer + RNase inhibitor for final resuspension. Add 18.6 ul RNase inhibitor to 1.5 ml nuclei buffer.
SECTION: Setup
7. Thaw components of 1 Parse Biosciences Nuclei Fixation v2 kit at room temperature, then place on ice.
SECTION: Setup
8. 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
9. Keep flash frozen tissue samples on dry ice until lysis.
SECTION: Tissue lysis and nuclei extraction
10. Drop whole 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. There should be 4 pieces: left and right ovary and left and right oviduct.
SECTION: Tissue lysis and nuclei extraction
11. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes).
SECTION: Tissue lysis and nuclei extraction
12. 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
13. Filter nuclei suspension through 30 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
14. Wash 30 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
15. Discard strainer and centrifuge the 4 ml nuclei suspension at 4C, 350g for 5 minutes. 
SECTION: Tissue lysis and nuclei extraction
16. Discard supernatant and resuspend nuclei pellet 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. In our experience, total number of nuclei from female gonads is around 4 million.
SECTION: Parse Nuclei Fixation
18. Add 250 uL Nuclei Fixation Solution and mix 3 times. Do not over-mix.
SECTION: Parse Nuclei Fixation
19. 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
20. 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
21. Incubate 3 minutes with nuclei on ice.
SECTION: Parse Nuclei Fixation
22. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. 
SECTION: Parse Nuclei Fixation
23. Centrifuge at 4C, 750g for 10 minutes.
SECTION: Parse Nuclei Fixation
24. Aspirate and discard supernatant.
SECTION: Parse Nuclei Fixation
25. Resuspend the samples that started with < 2 million nuclei in 150 ul Nuclei Buffer with RNase inhibitor without BSA and transfer to a labeled 1.5 ml tube. Do NOT filter, will lose too much volume. For samples starting with > 2 million nuclei, resuspend in 300 ul Nuclei Buffer and filter.
SECTION: Parse Nuclei Fixation
26. Count nuclei. Use a 1:11 dilution factor, e.g. 2 ul + 20 ul dye.
SECTION: Parse Nuclei Fixation
27. Add Nuclei DMSO: 2.5 ul into 150 ul samples and gently flick tubes to mix. One minute later, add another 2.5 ul and flick to mix, then after another minute add a final 2.5 ul for a total volume of 7.5 ul. Mix by gently pipetting 5x with a P200 set to 75 ul.
SECTION: Parse Nuclei Fixation
28. Split nuclei suspension into 2 aliquots of equal volume in labeled 1.5 ml tubes. (E.g. if resuspend with 150 ul, split nuclei suspension into 75 ul per tube.)
SECTION: Parse Nuclei Fixation
29. Place tubes in a Mr. Frosty at -80C. The next day, move tubes to boxes in -80C racks. 
SECTION: Tissue lysis and nuclei extraction
17. Count nuclei. Use 1:6 dilution factor, 4 ul + 20 ul dye.
SECTION: Storage of leftover nuclei
30. Move remaining nuclei in RSB on ice to labeled 2 ml tubes.
SECTION: Storage of leftover nuclei
31. Spin 750g, 4C, 5 minutes.
SECTION: Storage of leftover nuclei
32. Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80C.