TITLE: Nuclear Isolation of Post-Mortem Brain Tissue for snRNAseq
AUTHORS: Jonathan W Brenton, Carlo Sala Frigerio, Mina Ryten, Regina H Reynolds, e.gustavsson, Kylie Montgomery

[DESCRIPTION]
This protocol is used to isolate nuclei from frozen brain tissue for single nuclear RNA sequencing using 10x Genomics GEM isolation using the Chromium accessory and Single Cell 3ʹ Reagent Kits.

It is adapted from a protocol designed by Carlo Sala Frigerio.

The nuclei are homogenised by hand, run through a density gradient, resuspended, counted and diluted to an appropriate concentration to run in the Chromium.

This protocol is designed to be continued with the Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) to isolate nuclei in GEMs.

[STEPS]
SECTION: Buffer Preparation on ice for 6 samples (amount prepared for 7 to allow for waste)
3. ODN 
  
       ODNV (µl)Stock conc Final concKCl1342.68752000mM150mMMgCl2537.0751000mM30mMTris pH 7,4 (Sigma)1074.151000mM60mMSucrose8951.25500mM250mMNuclease free water5997.3375Total Volume17902.5 

 
 
SECTION: Buffer Preparation on ice for 6 samples (amount prepared for 7 to allow for waste)
4. 29% Cushion
 
       29% CushionV (µl)Stock conc Final concOptiprep1522560%29%ODN16275Total Volume315004500per sample 

 
 
SECTION: Buffer Making for 6 samples (7x made to allow for waste)
1. Prepare buffers from stocks, keep  on ice .
SECTION: Buffer Preparation on ice for 6 samples (amount prepared for 7 to allow for waste)
2. Gradient Medium

       GM (Gradient Medium)V (µl)Stock conc Final concCaCl21051000mM5mMOptiprep1750060%50%MgCl2631000mM3mMTris pH 7,42101000mM10mMPMSF4250mM0.1mMβ-mercaptoethanol42050mM1mMNuclease free water2660.0Total Volume210003000per sample 

 
 
4. 
1. 
2. 
SECTION: Homogenisation
5. Cool down ultracentrifuge rotor and adapters to 4C (alternatively rotors and adapters can be left overnight in fridge). If it doesn't cool quickly with normal function: set to spin at 1000rpm at 4C.
SECTION: Homogenisation
6. Rinse homogenizers with HB buffer on ice. Perform a few strokes in buffer and empty.
SECTION: Homogenisation
7. Add 1ml HB to douncer on ice
SECTION: Homogenisation
8. Take out and weigh sample amount by putting douncer on scale - Aim for ~100 mg. A range of 80-150mg has performed well.
SECTION: Homogenisation
10. Homogenise: ~20 strokes with pestle A and then ~80 strokes with pestle B; try to keep on ice as much as possible. If meninges present following 10 strokes with pestle B remove with forceps as this can add a lot of resistance. Clean forceps in dH20 water, wipe clean and then ethanol spray and dry.
SECTION: Homogenisation
9. 
SECTION: Buffer Preparation on ice for 6 samples (amount prepared for 7 to allow for waste)
1. Homogenisation Buffer
 
       HB (Homogenisation Medium)V (µl)Stock concFinal concSucrose17920.0500mM320mMCaCl2140.01000mM5mMMg Acetate168.0500mM3mMTris pH 7,4 (Sigma)280.01000mM10mMEDTA5.6500mM0.1mMNP40280.010%0.1%Digitonin56.05%0.01%PMSF56.050mM0.1mMβ-mercaptoethanol560.050mM1mMNuclease free water8534.4Total Volume28000.04000per sample 

 
 
SECTION: Buffer Making for 6 samples (7x made to allow for waste)
1.1. Homogenisation Buffer (HB)
      HB (Homogenisation Medium)V (µl)Stock concFinal concSucrose17920.0500mM320mMCaCl2140.01000mM5mMMg Acetate168.0500mM3mMTris pH 7,4 (Sigma)280.01000mM10mMEDTA5.6500mM0.1mMNP40280.010%0.1%Digitonin56.05%0.01%PMSF56.050mM0.1mMβ-mercaptoethanol560.050mM1mMNuclease free water8534.4Total Volume28000.04000per sample

 
 


SECTION: Buffer Making for 6 samples (7x made to allow for waste)
1.2. Gradient Medium

      GM (Gradient Medium)V (µl)Stock conc Final concCaCl21051000mM5mMOptiprep1750060%50%MgCl2631000mM3mMTris pH 7,42101000mM10mMPMSF4250mM0.1mMβ-mercaptoethanol42050mM1mMNuclease free water2660.0Total Volume210003000per sample

 
 
SECTION: Buffer Making for 6 samples (7x made to allow for waste)
1.3. ODN
      ODNV (µl)Stock conc Final concKCl1342.68752000mM150mMMgCl2537.0751000mM30mMTris pH 7,4 (Sigma)1074.151000mM60mMSucrose8951.25500mM250mMNuclease free water5997.3375Total Volume17902.5

 
 
SECTION: Buffer Making for 6 samples (7x made to allow for waste)
1.4. 29% Cushion
 
      29% CushionV (µl)Stock conc Final concOptiprep1522560%29%ODN16275Total Volume315004500per sample

 
 
SECTION: Homogenisation
2. Rinse douncers with some HB buffer – leave these on ice too.

SECTION: Homogenisation
3. Add 1 mL  HB to douncer.

SECTION: Homogenisation
4. Put douncer on scale in any support (falcon tube holder was used here), zero the scale and add sample to douncer. Aim for between 80-150mg tissue. ~100 mg of tissue works well.

SECTION: Homogenisation
5. Homogenise:~20 strokes with pestle A and then~80 strokes with pestle B; try to keep on ice as much as possible. If meninges present following 10 strokes with pestle B remove with forceps as this can add a lot of resistance. Clean forceps in dH20 water, wipe clean and then spray with 70% ethanol and wipe and air dry.

SECTION: Homogenisation
7. For each sample transfer ~ 1 mL homogenate (everything in douncer) to 15mL falcon and add 1650 µL  HB (total V = 2.65 mL). Mix by inverting as well as pipetting. All on ice.

SECTION: Homogenisation
8. Add and mix 2.65 mL (0.883 mL x 3) Gradient medium to sample (total V = 5.30 mL). Mix by inverting gently as well as pipetting. All on ice.

SECTION: Homogenisation
6. Keep douncer with homogenised sample on ice for  and repeat steps 3-6 for other samples.
SECTION: Gradient Preparation and Isolation by Centrifugation
9. Add 4 mL of optiprep cushion to an ultracentrifuge tube on ice for each sample.

SECTION: Gradient Preparation and Isolation by Centrifugation
10. Layer 5.25 mL sample on top of cushion. To do this slowly add homogenate/gradient medium mixture just above the top of the optiprep cushion and keeping the pipette tip against the wall of the ultracentrifuge tube. One way to do this is to use a P200 first to layer 200 µL-400 µL of sample on top of the cushion and the proceed with a P1000 to layer additional mLs of sample. Keep thumb under plunger of pipette to make sure addition of sample is gradual and is not pushed to the bottom of the gradient. Try and keep the bottom of the tube touching the ice as much as possible to keep the tube cool.

SECTION: Gradient Preparation and Isolation by Centrifugation
11. Check that sample's weight in ultracentrifuge tube is balanced (do in pairs – ie samples facing each other in ultracentrifuge should be equal in weight), in case adjust weight with HB.
SECTION: Gradient Preparation and Isolation by Centrifugation
13. Spin samples in SW41Ti 7700 rpm 30 min  4 °C - keep all tube holders in the machine even if empty and keep positions matched, ie holder 1 is in position 1. The ultracentrifuge should be pre-cooled to 4 °C  prior to adding the samples.

SECTION: Gradient Preparation and Isolation by Centrifugation
12. Add samples to ultracentrifuge buckets.
SECTION: During Ultracentrifugation
14. Take out 10X beads from -80 °C  and GEM kit from-20 °C. Prepare Template Switch Oligo as per 10x instructions if needed.

SECTION: During Ultracentrifugation
15. Prepare the resuspension buffer:
SECTION: During Ultracentrifugation
15.1. RNAseq Resuspension Buffer (for 6 samples + 1 for waste). Prepare and keep on ice.
 
      RNAseq Resuspension BufferV (uL)stockfinalPBS ( w/o Ca and Mg)9464BSA168010%1.5%RNase IN (Promega Purple cap)5640u/uL0.2u/uLfinal volume11200

 
 
SECTION: Resuspension and filtering
16. Gently take away supernatant using plastic Pasteur or P1000, then P200 Gilson. Nuclei will be a white smear at the bottom of the tube, the tube will gain condensation quickly so can wipe it to get a better look at the nuclei at the end and try and hold up the tube to a dark surface to get a look at the nuclei (although not all can be seen). Keep tube steady  and try not to mix and shake, which may resuspend the nuclei. Remove as much supernatant as possible by pipetting from the surface of the liquid and following the layer down to the lip of the ultracentrifuge tube. At this point remove as much as possible before nuclei start to come along with supernatant. Usually 200 µL - 500 µL remains at the bottom of tube. 

When in doubt leave supernatant as not to decrease yield or keep supernatant in a separate tube and filter as a separate sample that could be combined later in case of low yield. However, the vast majority of nuclei will be present at the bottom of the tube and this typically doesn't add many nuclei.

SECTION: Resuspension and filtering
17. Add in 200 µL of suspension buffer, transfer to chilled 1.5 or 2mL LoBind tube (either protein or DNA) on ice. All further steps should also be done  on ice and nuclei/samples should be kept cool as much as possible to prevent nuclei clumping.

SECTION: Resuspension and filtering
18. Wash out ultracentrifuge tube with further 200 µL of suspension buffer, pool in the LoBind tube  on ice.

SECTION: Resuspension and filtering
19. Add a further 400 µL of suspension buffer to the LoBind tube on ice (800 µL total suspension buffer).

SECTION: Resuspension and filtering
20. Mix gently by pipetting and filter through a filter cap Falcon tube and add to a new 1.5 or 2ml LoBind tube (protein or DNA) on ice.
SECTION: Resuspension and filtering
21. Keep on ice until the other samples have been resuspended and filtered.
SECTION: Resuspension and filtering
22. Centrifuge  500 rcf for 5 min and at 4 °C.

SECTION: Resuspension and filtering
23. Nuclei can now be seen, after wiping away condensation, as a white smear on the side of the tube in the DNA LoBind tubes or more of a pellet if using protein LoBind tubes. Remove supernatant and resuspend nuclei in 400 µL resuspension buffer by pipetting.

SECTION: Resuspension and filtering
24. Filter through a filter-cap Falcon tube.
SECTION: Resuspension and filtering
25. Transfer to LoBind Eppendorf on ice and centrifuge  500 rcf for 5 min and at 4 °C.

SECTION: Resuspension and filtering
26. Remove supernatant and resuspend nuclei in 400 µL (200 µL if nuclei pellet looks small in Lo Bind tube) resuspension buffer by pipetting. 

SECTION: Resuspension and filtering
27. Filter through a filter-cap Falcon tube. Keep on ice.
SECTION: Nuclei Counting
28. Count each aliquot with Luna FL (18 µL sample + 2 µL Acridine Orange/Propidium Iodide). Add 10 µL to slide. Save images and counts.

SECTION: Nuclei Counting
29. Create a new RNAseq aliquot of 1000 nuclei/uL in 80 µL by diluting original sample.

SECTION: Nuclei Counting
30. Count diluted sample and record number (save files if needed but adds time for each sample).
SECTION: Nuclei Counting
31. Round count to nearest 100 and use this when loading the chip. Ie if one sample has concentration of 1100 and another 900 use two different cell concentrations in 10x protocol to load rather than rediluting sample to achieve 1000 cells.