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

[DESCRIPTION]
This protocol describes isolation of nuclei from 10 week old mouse hypothalamus and pituitary gland (tissue ID: 01) from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for 1. single nucleus RNA-seq using the Parse Biosciences protocol (Split-seq) and 2. single nucleus RNA-seq + ATAC-seq using the SHARE-seq protocol. 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 are 2 aliquots of fixed single-nucleus suspensions for Parse per each of the 8 samples at >= 2,500 nuclei/ul, and 1 fixed nuclei pellet pooled across all 8 strains for SHARE-seq, all stored at -80C.

The first part of the protocol describes tissue lysis and nuclei extraction using Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. When nuclei are extracted and counted, we determine whether we have enough to fix for Split-seq and SHARE-seq and set aside 2-4 million and 1 million, respectively. Ideally, the second and third parts of this protocol are performed in parallel by at least two technicians to save time. The second part describes nuclei fixation using Parse Biosciences Evercode Nuclei Fixation Kit with v2 reagents (see attachment for original version). The third part describes nuclei fixation using a modified version of the SHARE-seq fixation protocol (see attachment for original version). Any remaining nuclei are flash-frozen as a dry pellet and stored at -80C.

[GUIDELINES]
We recommend using a 5 ml pipette for aspirations and resuspensions > 1 ml.
Record everything in the IGVF spreadsheet, “Samples into experiment” tab.
When possible, after nuclei isolation and during the first round of counting, 2 technicians should continue on with Parse fixation after establishing the volume needed for roughly 4 million nuclei per sample (4 samples processed per technician). The remaining technician should determine the exact volume needed for 1 million cells and proceed with SHARE-seq fixation. Parallel fixation saves about an hour of time.

[STEPS]
SECTION: Setup
1. Coat SHARE-seq nuclei prep tubes with BSA. Fill 8 1.5 ml tubes with 1.5 ml 1% BSA-DEPC and incubate for 30 minutes. After incubation, aspirate BSA solution and dry for 30 minutes. Store at 4C.
SECTION: Setup
2. Label tubes.
SECTION: Setup
3. Pre-chill centrifuge to 4C.
SECTION: Setup
4. Prepare ice buckets.
SECTION: Setup
5. Prepare 35 mL lysis buffer in a 50 ml conical tube on ice. 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 RSB in a 50 ml conical tube on ice. Add RNase inhibitor the day of the experiment.
SECTION: Setup
7. 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
8. Prepare 2.5 ml nuclei buffer + RNase inhibitor for final resuspension. Add 31.5 ul RNase inhibitor to 2.5 ml nuclei buffer.
SECTION: Setup
9. Prepare 15 ml SHARE-RSB in a 50 ml conical tube at room temperature. To SHARE-RSB, add 30 ul digitonin, 37.5 ul Enzymatics RI, 18.75 ul SUPERase RI, and 150 ul yeast tRNA fresh the day of the experiment.
SECTION: Setup
10. Thaw components of 2 Parse Biosciences Nuclei Fixation v2 kits at room temperature, then place on ice.
SECTION: Setup
11. 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
12. Keep flash frozen tissue samples on dry ice until lysis.
SECTION: Tissue lysis and nuclei extraction
13. 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.
SECTION: Tissue lysis and nuclei extraction
14. Run the gentleMACS Program 4C_nuclei_1 on the Octo Dissociator (~5 minutes).
SECTION: Tissue lysis and nuclei extraction
15. 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
16. 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
17. 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
18. Discard strainer and centrifuge the 4 ml nuclei suspension at 4C, 350g for 5 minutes. 
SECTION: Tissue lysis and nuclei extraction
19. Discard supernatant and resuspend nuclei pellet in 3 ml RSB.
SECTION: Tissue lysis and nuclei extraction
20. Filter nuclei suspension through 30 um MACS SmartStrainer into a 5 ml tube.
SECTION: Tissue lysis and nuclei extraction
21. Count nuclei. Use 1:3 dilution factor, 10 ul + 20 ul dye.
SECTION: Parse nuclei fixation
22. Set aside 2-4 million nuclei in RSB in a new 1.5 ml tube and spin down at 4C, 350g for 5 minutes.
SECTION: Parse nuclei fixation
23. 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
24. Add 250 uL Nuclei Fixation Solution and mix 3 times. Do not over-mix.
SECTION: Parse nuclei fixation
25. 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
26. 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
27. Incubate 3 minutes with nuclei on ice.
SECTION: Parse nuclei fixation
28. Add 4 ml Nuclei Neutralization Solution and invert the tube once to mix. 
SECTION: Parse nuclei fixation
29. Centrifuge at 4C, 750g for 10 minutes.
SECTION: Parse nuclei fixation
30. Aspirate and discard supernatant.
SECTION: Parse nuclei fixation
31. Resuspend the samples in 200 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
32. Count nuclei. Use 1:11 dilution factor, e.g. 2 ul + 20 ul dye.
SECTION: Parse nuclei fixation
33. Add Nuclei DMSO: 3.3 uL and gently flick tubes to mix. One minute later, add another 3.3 uL and flick to mix, then after another minute add a final 3.3 uL for a total volume of 9.9 uL. Mix by gently pipetting 5x with a P200 set to 100 ul.
SECTION: Parse nuclei fixation
34. Split nuclei suspension into 2 labeled tubes, 100 ul per tube.
SECTION: Parse nuclei fixation
35. Place tubes in a Mr. Frosty at -80C. The next day, move tubes to boxes in -80C racks. 
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 on ice to 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.