TITLE: Nuclei isolation, immunostaining, and Fluorescence-activated nuclei sorting (FANS) for Smart-Seq2
AUTHORS: Ester Kalef-Ezra, Dominic Horner, George Morrow, Vanda Knitlhoffer, Andy Goldson, Iain Macaulay, Yanping Guo, Christos Proukakis

[DESCRIPTION]
This protocol describes the steps for extracting nuclei from human postmortem brain samples, immunofluorescence, and nuclei sorting (FANS) for single-nucleus RNA-seq using the Smart-Seq2 method. 
We have used it to isolate nuclei from human brain samples (such as the cingulate cortex), but it can be adapted for nuclei from different body areas, cell culture materials, and/or different species.

[GUIDELINES]
Unless otherwise indicated, all the reagents must be kept at 4 °C and all the steps in the protocol must be performed at 4 °C. 


[STEPS]
SECTION: Nuclei isolation from human post-mortem brain tissue
1. Preparation:

UV-treat 96-well plates prior to use.

 

 

 

 

SECTION: Nuclei isolation from human post-mortem brain tissue
2. Pre-weight tubes are needed for tissue scaling.
SECTION: Nuclei isolation from human post-mortem brain tissue
3. Clean pestles the day before with 0.2 Molarity (M) NAOH, 10% Presept and place them in a falcon tube containing RNase AWAY Decontamination Reagent. 
SECTION: Nuclei isolation from human post-mortem brain tissue
4. Incubate the pestles with the RNase AWAY O/N @Room temperature. 
SECTION: Nuclei isolation from human post-mortem brain tissue
5. Prepare PBS (1x, RNase-free) and, Sorting Buffer (1x PBS + 5 millimolar (mM)EDTA).

SECTION: Experimental steps:
6. Clean Human Tissue handling hood with 0.2 Molarity (M) NaOH, 10% Presept, 70% EtOH and dH2O (nuclease-free).
SECTION: Experimental steps:
7. Prepare the Lysis Buffer in the materials section.
SECTION: Experimental steps:
8. Distribute 2 µL Lysis Buffer in each well of the plates needed for sorting.
SECTION: Experimental steps:
9. Wash the pestles thoroughly with MiliQ-H2O and then with dH2O (RNase-free) and let them air-dry on a clean wipe tissue before use. Once dried, place them in a clean wipe tissue in a clean container (e.g., falcon tube or sealed bag) and put them in the fridge to pre-cool. 

 

SECTION: Experimental steps:
10. Clean human handling hood with 0.2 Molarity (M) NaOH, 10% Precept, 70% EtOH, RNaseZap.
SECTION: Experimental steps:
11. Transfer all materials needed, carefully handle human brain samples in a human handling hood, and wash them with RNaseZap.
SECTION: Experimental steps:
12. Prepare 5% BSA in PBS (1x) and store on ice until use, e.g., for 1 sample.
SECTION: Experimental steps:
13. Add RNasin Plus Ribonuclease Inhibitor to Buffer A and B prior touse to afinal concentration of 0.2 U/µl , e.g., for 1 sample:
 
Buffer A: 5 µL RNasin Plus Ribonuclease Inhibitor in 995 µL Buffer A
Buffer B: 5 µL RNasin Plus Ribonuclease Inhibitor in 995 mL Buffer B


SECTION: Experimental steps:
14. Remove tissue from -80 °C freezer and transfer it to the human tissue handling hood on dry-ice.
SECTION: Experimental steps:
15. Cut small brain pieces of tissue of interest in pre-weighed 1.5 ml Eppendorf tubes.
SECTION: Experimental steps:
16. Weigh the tissue in a scale aiming for 15 mg-30 mg of each tissue/donor depending on the downstream needs.

 

SECTION: Experimental steps:
17. Add 200 µL cold Buffer A (containing 0.2 U/µl RNasin Plus Ribonuclease Inhibitor) in each tube containing tissue and place it on wet ice. 

 

SECTION: Experimental steps:
18. Homogenize the tissue using the pestle (pre-chilled) provided by grinding gently with twisting force for 50-60 times.

 

 

SECTION: Experimental steps:
19. Add 500 µL cold Buffer A (containing 0.2 U/µl RNasin Plus Ribonuclease Inhibitor) to the tube and continue to grind for 20-30 times. 
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20. Incubate the tube on ice for 5 min and carefully transfer homogenate to a filter (column) in collection tube (avoid larger debris that sinks to the bottom of the tube). 
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21. Incubate the tube with the cap open at -20 °C for 7 min.

 

SECTION: Experimental steps:
22. Cap the filter and immediately centrifuge at 13000 x g, 30 s, 4 °C.
SECTION: Experimental steps:
23. Discard the filter (column) and resuspend the pellet by pipetting up and down gently for 10-20 times. 

 

 

 

SECTION: Experimental steps:
24. Centrifuge at 600 x g, 5 min, 4 °C. 
SECTION: Experimental steps:
25. Pour out the supernatant and resuspend the pellet in 200 µL PBS with 5% BSA that will be overlaid on top of Buffer B (containing 0.2 U/µl RNasin Plus Ribonuclease Inhibitor) in the next step.

 

SECTION: Experimental steps:
26. Add 1 mL cold Buffer B (containing 0.2 U/µl RNasin Plus Ribonuclease Inhibitor) to a 1.5 ml Eppendorf tube. 
 

SECTION: Experimental steps:
27. Carefully overlay the 200 µl nuclear suspension from step 17 on top of Buffer B by slowly expelling the nuclear suspension against the wall of the tube.
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28. Centrifuge the tube at 1000 x g, 10 min, 4 °C.

 

 

 

SECTION: Experimental steps:
29. Carefully remove the milky layer by withdrawing it into a 1 ml pipette tip and discarding the rest of the supernatant.
SECTION: Experimental steps:
30. Pour out the remaining Buffer B, leaving 50 µL in the bottom of the ultracentrifuge tube (as it contains the nuclear fraction).
SECTION: Section 2: Nuclei immunostaining
31. Prepare Blocking Buffer: 0.8% BSA + 0.2 U/µl RNasin in 10X PBS, pH 7.4 (1x), e.g. for 2 ml:
 
 

 

SECTION: Section 2: Nuclei immunostaining
32. Resuspend the pellet in 450 µl (or a volume of choice depending on the initial tissue input) of cold Blocking Buffer and resuspend by pipetting up and down gently 5 times. 
 

SECTION: Section 3. Nuclei sorting (FANS)
46. Before single-cell sorting, use Accudrop beads for a test sort to evaluate the plate's position and ensure the sorted cells will be deposited into each well accurately in the middle. 
SECTION: Section 3. Nuclei sorting (FANS)
47. As an extra layer of assessment for accurate sorting, assess plate positioning with colorimetric method.
SECTION: Section 3. Nuclei sorting (FANS)
47.3. To run the test:

Aliquot 2 µL of TMB substrate (fridge in the FACS room) into each well of the test plate.
SECTION: Section 3. Nuclei sorting (FANS)
47.4. Sort a single bead into a whole plate (or wells needed).
SECTION: Section 3. Nuclei sorting (FANS)
47.5. Once the sort is completed, immediately seal the plate and centrifuge (500 x g, 1 min) and wait 5 min-10 min and count the number of wells that have turned blue.

 

 

SECTION: Section 3. Nuclei sorting (FANS)
48. Spin the sorting plates and arrange plate orientation.

 

 

Select gating parameters to isolate the singlets from the overall detected particles by selecting forward (FSC) and side scatter (SSC), FCS single cell gate, and SSC single cell gate. 
Then select the nuclei by their DAPI expression. 
From the nuclei population (DAPI+), apply further gating parameters based on the antibodies used.

 

 

SECTION: Section 3. Nuclei sorting (FANS)
49. Centrifuge for 10 sat low speed the collection plates to ensure reagent at the bottom. 
SECTION: Section 3. Nuclei sorting (FANS)
50. Sort single-nuclei of interest into the centrifuged 96-well collection plates.
SECTION: Section 3. Nuclei sorting (FANS)
51. After sorting, seal carefully the plates with adhesive plate foil and a seal and place on ice. 
SECTION: Section 3. Nuclei sorting (FANS)
52. Immediately centrifuge briefly at 500 x g, 1 min, 4 °C.
SECTION: Section 3. Nuclei sorting (FANS)
53. Place each plate in an individually sealed bag (dry-ice resistant).
SECTION: Section 3. Nuclei sorting (FANS)
54. Place immediately on dry-ice and transfer to the lab if needed.
SECTION: Section 3. Nuclei sorting (FANS)
55. Store the plate(s) -70 °C until further use and transfer the plates on dry-ice, if necessary, until single-cell Smart-Seq2.
SECTION: Section 4. Single-nucleus Smart-Seq2
56. Smart-Seq2 is performed according to Picelli et al. 2014 (Nature Protocols) with the following modifications:

o	cDNA is amplified with 25 cycles of PCR.
o	oligo-dT30VN, template-switching oligonucleotide (TSO), and IS PCR primers are modified by 5′ biotinylation (Zeisel et al. 2015, Science) (all ordered from IDT).

 


 

SECTION: Section 4. Single-nucleus Smart-Seq2
57. 0.6X SPRI-bead clean-up is performed on the cDNA to minimize the presence of short fragments (e.g., primer-dimers) using Biomek NX (Beckman Coulter) liquid handling robot.
SECTION: Section 4. Single-nucleus Smart-Seq2
58. cDNA samples are normalized to0.2 ng/µL based on an average concentration of 11 samples ran on the bioanalyzer.
SECTION: Section 4. Single-nucleus Smart-Seq2
59. Library construction is performed using the Nextera XT sample prep kit (Illumina), a miniaturized protocol involving 12 cycles of PCR.
SECTION: Section 4. Single-nucleus Smart-Seq2
60. Libraries are prepared in 384-well PCR plates. The I.DOT (Dispendix) instrument is used to array reagents and indices.
SECTION: Section 4. Single-nucleus Smart-Seq2
61. Libraries were pooled prior to library clean-up using the Mosquito (SPT Labtech), and 0.8X SPRI-bead clean-up was performed on the 96-plex “plate pools” by hand. “Plate pools” were pooled equimolarly after QC using Bioanalyzer and qPCR.

 

SECTION: Section 4. Single-nucleus Smart-Seq2
62. The pooled library is sequenced in an Illumina sequencer. 

 

SECTION: Section 3. Nuclei sorting (FANS)
47.1. Add 1 mL of dH2O into the vial of powder HRP, and dissolve (this stock is 10x concentrated as compared to working solution). 
SECTION: Section 3. Nuclei sorting (FANS)
47.2. Make a working solution ( 1 mg/mL) by:

To get 2 mL:

200 µL stock HRP (10 mg/mL) 
1800 µL diH2O
2 drops of Accudrop beads

Keep in the fridge.
SECTION: Section 2: Nuclei immunostaining
33. Separate the samples into 2 tubes each:

a.	With Abs: 500 µl
b.	Negative Control (with isotype control and/or without antibodies): remaining nuclei supplemented with Blocking Buffer to have a final volume 200 µl.

 

SECTION: Section 2: Nuclei immunostaining
34. Incubate the nuclei in Blocking Buffer for 30 min @4 °C in a rotating wheel or in falcon tubes in a tube roller.
SECTION: Section 2: Nuclei immunostaining
35. Add antibodies directly to the Blocking Buffer.
SECTION: Section 2: Nuclei immunostaining
36. Add DAPI (the 1/10.000, e.g. add 5 µl of DAPI diluted 1/100 in PBS 1x) to all nuclei.

 

SECTION: Section 2: Nuclei immunostaining
37. Incubate all samples for at least 30 min at 4 °C in a rotating wheel or falcon tubes in a tube roller.
SECTION: Section 2: Nuclei immunostaining
38. Pellet nuclei at 800 x g, 10 min, 4 °C.

 

 

SECTION: Section 2: Nuclei immunostaining
39. Carefully discard the supernatant, leaving ∼50 µl of buffer above the pellet.
SECTION: Section 2: Nuclei immunostaining
40. Resuspended in pre-chilled Blocking Buffer (same volume as before) by gently pipetting up and down 5 times.
SECTION: Section 2: Nuclei immunostaining
41. Re-pellet nuclei at 800 x g, 10 min, 4 °C and discard the supernatant.
SECTION: Section 2: Nuclei immunostaining
42. Wash nuclei again with Blocking Buffer.
SECTION: Section 2: Nuclei immunostaining
43. Pellet nuclei at 800 x g, 10 min, 4 °C and carefully discard the supernatant.
SECTION: Section 2: Nuclei immunostaining
44. Resuspend nuclei in 600 µl (or volume of interest depending on the input tissue) pre-chilled Blocking Buffer and proceed to filtering using Flowmi Cell Strainers in clean 1.5 mL Eppendorf tubes.
SECTION: Section 2: Nuclei immunostaining
45. Transfer the nuclei on ice to sorting facility on ice and perform the sorting as soon as possible.