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

[DESCRIPTION]
This protocol describes isolation of nuclei from 10 week old mouse kidneys 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.

[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 lysis buffer in a 50 ml conical tube on ice. Distribute 2.5 ml into 8 gentleMACS C Tubes on ice. Add RNase inhibitor to the lysis buffer aliquot the day of the experiment.
SECTION: Setup
5. Prepare RSB in a 50 ml conical tube on ice. Add NEB RNase inhibitor the day of the experiment.
SECTION: Setup
6. Prepare NB + BSA + RNase inhibitor. Add RNase inhibitor included in Parse Biosciences fixation kit the day of the experiment.
SECTION: Setup
7. Prepare 2.5 ml Nuclei Buffer + RNase inhibitor for final resuspension. Add 31.5 ul Parse RNase inhibitor to 2.5 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. Keep flash frozen tissue samples on dry ice until lysis.
SECTION: Tissue lysis and nuclei extraction
11. Drop whole frozen tissue into a chilled gentleMACS C Tube with 2.5 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 2 kidneys.
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 4.5 ml nuclei suspension at 4C, 350g for 5 minutes. 
SECTION: Tissue lysis and nuclei extraction
17. Discard supernatant and resuspend nuclei pellet in 3 ml RSB. Filter nuclei suspension through 30 um MACS SmartStrainer into a 5 ml tube.
SECTION: Tissue lysis and nuclei extraction
18. Take 200 ul and add to a 5 ml tube that contains another 1.8 ml RSB for a 1:10 dilution (same as male gonads and similar to liver protocols).
SECTION: Tissue lysis and nuclei extraction
19. Count nuclei. Use 1:11 dilution factor, 2 ul + 20 ul dye.
SECTION: Tissue lysis and nuclei extraction
20. Centrifuge 4 million nuclei at 4C, 500g for 5 minutes and remove supernatant.
SECTION: Tissue lysis and nuclei extraction
21. Resuspend pellet in 750 ul NB-BSA + RNase inhibitor.
SECTION: Parse nuclei fixation
22. Filter the nuclei suspension (750 ul) NB-BSA + RNase inhibitor through a 40 um strainer (provided in Parse Biosciences kit) into a new 5 ml tube.
SECTION: Parse nuclei fixation
23. Add 250 uL Nuclei Fixation Solution and mix 3 times. Do not over-mix.
SECTION: Parse nuclei fixation
24. 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
25. 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
26. Incubate 3 minutes with nuclei on ice.
SECTION: Parse nuclei fixation
27. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. 
SECTION: Parse nuclei fixation
28. Centrifuge at 4C, 750g for 10 minutes.
SECTION: Parse nuclei fixation
29. Aspirate and discard supernatant.
SECTION: Parse nuclei fixation
30. Resuspend the samples in 300 ul Nuclei Buffer with RNase inhibitor without BSA and filter through a 40 um filter into a labeled 1.5 ml tube.
SECTION: Parse nuclei fixation
31. Count nuclei. Use a 1:11 dilution factor, e.g. 2 ul + 20 ul dye.
SECTION: Parse nuclei fixation
32. Add Nuclei DMSO: For 300 ul samples: add 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
33. Split nuclei suspension into 2 aliquots of equal volume in labeled 1.5 ml tubes.
SECTION: Parse nuclei fixation
34. Place tubes in a Mr. Frosty at -80C. The next day, move tubes to boxes in -80C racks. 
SECTION: Parse nuclei fixation
35. Move leftover nuclei suspension to labeled 2 ml tubes and spin at 4C, 750g for 5 minutes. Remove supernatant and flash-freeze nuclei in liquid nitrogen as dry pellets. Store at -80C.
SECTION: SHARE-seq nuclei fixation
36. Set aside 1 million nuclei for each of the 8 samples in RSB and spin down at 4C, 750g for 5 minutes.
SECTION: SHARE-seq nuclei fixation
37. Remove supernatant and resuspend nuclei pellet in 1 ml room temperature SHARE-RSB. Transfer tube to a room temperature rack.
SECTION: SHARE-seq nuclei fixation
38. At RT, add 13.34 ul of methanol-free formaldehyde (16% stock solution). Final concentration for nuclei: 0.2%. Close tube and nutate cells at RT for 5 minutes.
SECTION: SHARE-seq nuclei fixation
39. To quench fixation, per reaction, add 56.1 ul fresh 2.5M Glycine (0.94g per 5 ml stock), 50 ul of 1M Tris pH 8.0, 13.3ul of 7.5% BSA, and mix using a pipette. Incubate on ice for 10 minutes.
SECTION: SHARE-seq nuclei fixation
40. Spin 750g, 4C, 5 minutes. Gently remove supernatant.
SECTION: SHARE-seq nuclei fixation
41. Add 200 ul of SHARE-RSB and gently resuspend pellet. Store on ice until all samples are completed.
SECTION: SHARE-seq nuclei fixation
42. Pool 200 ul of resuspended nuclei from all 8 founders into 1 labeled 2 ml tube.
SECTION: SHARE-seq nuclei fixation
43. Spin 1,000g, 4C, 10 minutes. Gently remove supernatant. Remove all fluid and freeze at -80C as a dry pellet.
SECTION: Storage of leftover nuclei
44. Move remaining nuclei in RSB in 5 ml tubes on ice to new labeled 2 ml tubes.
SECTION: Storage of leftover nuclei
45. Spin 750g, 4C, 5 minutes.
SECTION: Storage of leftover nuclei
46. Remove all supernatant and flash-freeze nuclei as a dry pellet in liquid nitrogen. Store at -80C.