TITLE: Human post-mortem nuclei preparation and single-nucleus multiome DNA/RNA sequencing by ResolveOME
AUTHORS: Ester Kalef-Ezra, George Morrow, Yanping Guo, Christos Proukakis

[DESCRIPTION]
Here we describe a protocol for nuclei isolation, immunostaining and sorting (FANS: Fluorescence-activated nuclear sorting) for single-nucleus DNA/RNA-seq using ResolveOME (BioSkryB) method from human post-mortem brain samples. We have used it to isolate nuclei from human post-mortem substantia nigra tissue, but it can be adapted for nuclei from different body areas.

The ResolveOME protocol is generally designed to work with single live mammalian cells and here we have done minor modifications in order to use it for human post-mortem brain samples.

[GUIDELINES]
Critical note: Unless otherwise indicated, keep all the reagents and steps at 4 °C to protect DNA and RNA integrity. 
 

[STEPS]
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1. Experimental steps:

 

 

Day 1
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1.1. Clean pestles provided in the Nuclei Isolation Minute kit the day before with 0.2 Molarity (M) NAOH, 10% Presept and place them in a falcon tube containing RNase AWAY Decontamination Reagent. 

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1.2. Incubate the pestles in a 50 mL falcon tube containing RNase AWAY O/N @Room temperature.



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2. Day 2
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2.1. Wash pestles with MiliQ-H2O and then with dH2O (RNase-free), air-dry with a dry wipe and pre-cool at 4 °C.

 

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2.2. Clean carefully the PCR hood with 70% EtOH, dH2O, DNA AWAY and RNase Zap and place not opened sterile filtered pipette tips.
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2.3. Wipe the RNA lab pipettes with DNA AWAY and RNase Zap and place in the PCR hood.
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2.4. Place in the PCR hood Low Bind 96-well plates to be used for nuclei sorting.
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2.5. UV-treat the Low Bind 96-well plates and pipettes to be used for 20 min-30 min before use.

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2.6. Pre-weight 1.5 ml tubes need for tissue scaling.
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2.7. Prepare PBS (1x, RNase-free).
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2.8. Clean the human tissue handling hood with 0.2 Molarity (M) NaOH, 10% Presept, 70% EtOH and dH2O, DNA AWAY and RNase Zap.

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2.9. Transfer the pipettes, filtered tips in the human tisssue handling hood.
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2.10. Clean a pair of forceps with 0.2 Molarity (M) NaOH, 10% Presept, 70% EtOH and dH2O, DNA AWAY and RNase Zap and transfer it to the human tissue handling hood. 
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2.11. Place a small beaker with 0.2 Molarity (M) NaOH in the human tissue handling hood in order to use it to decontaminate the forceps and filtered tips after use.
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2.12. In the human tissue handling hood Prepare 5% BSA in PBS (1x) and store on ice until use, 
e.g. for 1 donor:
    
 

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2.13. Add RNasin Plus Ribonuclease Inhibitor to Buffer A and B prior to use to final 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

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2.14. Remove tissue from -80 °C freezer and transfer it to the human tissue handling hood on dry-ice.

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2.15. Cut small brain pieces of the tissue of interest in the pre-weighed 1.5 ml Eppendorf tubes.
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2.16. Weigh the tissue in a scale aiming for ~19 mg (aim for 15 mg-30 mg of each tissue/donor).

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2.17. 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. 

 

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2.18. Homogenize the tissue using a pre-chilled pestle by grinding gently with twisting force for 50-60 times.

 

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2.19. 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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2.20. Incubate the tube on ice for 5 min and carefully transfer homogenate to a filter (column) in collection tube (avoid larger debris that sink to the bottom of the tube).

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2.21. Incubate the tube with cap open at -20 °C for 7 min.

 


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

 

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2.24. Centrifuge at 600 x g, 5 min, 4 °C.

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2.25. 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.

 

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

 

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2.27. Carefully overlay the 200 µL nuclear suspension from    on top of Buffer B by slowly expelling the nuclear suspension against the wall of the tube.

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2.28. Centrifuge the tube at 1000 x g, 10 min, 4 °C.

 

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2.29. Carefully remove the milky layer by withdrawing it into a 1 ml pipette tip and discard the rest of the supernatant.
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2.30. Pour out the remaining Buffer B, leaving 50 µL in the bottom of the ultracentrifuge tube (as it contains the nuclear fraction). 

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3. Prepare Blocking Buffer: 0.8% BSA + 0.2 U/µl RNasin in 10X PBS, pH 7.4 (1x), 
e.g. for 2 mL: 
 
 Mix gently by inverting the tube 5-10 times and store it on ice until use.

 

 
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4. Resuspend the pellet in 500 µL (or volume of choice depending on the initial tissue input) of cold Blocking Buffer by pipetting up and down gently 5 times.

 

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5. Separate the samples into different tubes for nuclei with antibodies and negative control samples, e.g.:

A. Nuclei with PI and Olig2-AF647s: 500 µL.
B. Nuclei with PI and Isotype Control (IgG-AF647): 50 µL. Then supplement with Blocking Buffer to have final volume 200 µL.
C. Nuclei with PI, but no AF647: 50 µL. Then supplement with Blocking Buffer to have final volume 200 µL. 
D. Nuclei without PI or AF647: 50 µL. Then supplement with Blocking Buffer to have final volume 200 µL.
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6. Incubate the nuclei in Blocking Buffer for 30 min @4 °C on a plate shaker (tubes coved in parafilm, placed inside a falcon tube and the falcon tube on a plate shaker in the fridge).

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7. Add antibodies directly to the nuclei in Blocking Buffer.
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8. Incubate all samples for at least 30 min @4 °C on a plate shaker (tubes coved in parafilm and foil, placed inside a falcon tube and the falcon tube on a plate shaker in the fridge).

 

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9. Pellet nuclei at 800 x g, 10 min, 4 °C.

 

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10. Carefully discard supernatant leaving ~50 µL of buffer above the pellet.

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11. Resuspended in pre-chilled Blocking Buffer (same volume as before) by gently pipetting up and down 5 times.
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12. Re-pellet nuclei at 800 x g, 10 min, 4 °C and discard the supernatant.

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13. Wash nuclei again with Blocking Buffer.
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14. Pellet nuclei at800 x g, 10 min, 4 °C and carefully discard the supernatant.

 

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15. Resuspend nuclei to sort in 800 µL and the control nuclei in 200 µL Blocking Buffer with PI (or volume of interest depending on the input tissue).

 

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16. Re-label clean 1.5 ml low bind Eppendorf tubes.
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17. Filter the nuclei using Flowmi Cell Strainers and place the filtered nuclei in the pre-labelled clean 1.5 mL low binding Eppendorf tubes.
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18. Transfer the nuclei to the sorting facility on ice and perform the sorting as soon as possible.

 

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19. Before single-nucleus sorting, use Accudrop beads for a test sort to evaluate the position of the plate and to ensure the sorted cells will be deposited into each well accurately in the middle.
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20. Optional: As an extra layer of assessment for accurate sorting, assess plate positioning with colorimetric method. 

Adapted from: Rodrigues OR, Monard S. A rapid method to verify single-cell deposition setup for cell sorters. Cytometry A. 2016 Jun;89(6):594-600. doi: 10.1002/cyto.a.22865. PubMed PMID: 27144818
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20.1. Add 1 mL of dH2O into the vial of powder HRP, dissolve (this stock is 10x concentrated as compared to working solution).

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20.2. Make a working solution (1 mg/ml), e.g. For 2 mL: 200µl stock HRP (10 mg/ml) + 1800 µL diH2O + 2 drops of Accudrop beads. Store in the fridge.

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20.3. To run the test:

Aliquot 2 µL of TMB into each well of the test plate.
Sort a single bead into a whole plate (or wells needed).
Once the sort is completed, immediately seal the plate and centrifuge 500 x g, 1 min.
Wait 5 min-10 min and count the number of wells that have turned blue.

 

4. If successful deposition is achieved, proceed with sorting cells. If not successful, recalibrate the alignment and try again!

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21. Acquire small amounts of nuclei from all samples and perform a batch analysis to assess the sample quality and identify the targeted populations for sorting. 

 

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22. 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.
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23. Select the human post-mortem nuclei by their PI expression.
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24. From the nuclei population (PI+), apply further gating parameters based on the antibodies used.

Figure 1. Example of the gating strategy of human post-mortem samples. 

A. Nuclei with Olig2-AF647 and PI
 

 



 

B. Nuclei with isotype AF647 control and PI
 

   C. Nuclei with PI but no AF647
 

D. Nuclei without PI (no nuclei selection) or AF647
 

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25. Centrifuge for 10 s at low speed the collection plates to ensure reagent at bottom and place them on ice.

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26. Sort single-nuclei of interest into the centrifuged 96-well collection plates that are placed in a pre-cooled plate holder.
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27. After sorting, seal immediately seal the plates with VersiCap Mat cap strips and pre-labelled plate sealers.
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28. Immediately centrifuge briefly at 500 x g, 1 min, 4 °C.

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29. Place each plate in individually sealed bag.
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30. Place immediately on dry-ice and transfer to the lab, if needed.
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31. Stored the plate(s) -70 °C until further use.

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32. Follow the ResolveOME kit according to BioSkryB guidelines (BioSkryB TAS-074.1).