Nuclear Isolation of Post-Mortem Brain Tissue for snRNAseq

Prepare buffers from stocks, keep On ice .

Homogenisation Buffer (HB)

A	B	C	D	E	F
HB (Homogenisation Medium)	V (µl)	Stock conc		Final conc
Sucrose	17920.0	500	mM	320	mM
CaCl2	140.0	1000	mM	5	mM
Mg Acetate	168.0	500	mM	3	mM
Tris pH 7,4 (Sigma)	280.0	1000	mM	10	mM
EDTA	5.6	500	mM	0.1	mM
NP40	280.0	10	%	0.1	%
Digitonin	56.0	5	%	0.01	%
PMSF	56.0	50	mM	0.1	mM
β-mercaptoethanol	560.0	50	mM	1	mM
Nuclease free water	8534.4
Total Volume	28000.0	4000	per sample

Resuspend Digitonin first.For Digitonin: If a precipitate forms in the 5% Digitonin solution, we performed the recommend steps in the manual. This requires heating the solution at 95 degrees C for 5 mins and vortexing slowly to dissolve the precipitate. Cool to room temperature prior to use. The 5% Digitonin solution stays in solution at room temperature for up to 1 week.Although in practice this tends to precipitate after a day so most likely this will need reheating after a day or two.

Gradient Medium

A	B	C	D	E	F
GM (Gradient Medium)	V (µl)	Stock conc		Final conc
CaCl2	105	1000	mM	5	mM
Optiprep	17500	60	%	50	%
MgCl2	63	1000	mM	3	mM
Tris pH 7,4	210	1000	mM	10	mM
PMSF	42	50	mM	0.1	mM
β-mercaptoethanol	420	50	mM	1	mM
Nuclease free water	2660.0
Total Volume	21000	3000	per sample

ODN

A	B	C	D	E	F
ODN	V (µl)	Stock conc		Final conc
KCl	1342.6875	2000	mM	150	mM
MgCl2	537.075	1000	mM	30	mM
Tris pH 7,4 (Sigma)	1074.15	1000	mM	60	mM
Sucrose	8951.25	500	mM	250	mM
Nuclease free water	5997.3375
Total Volume	17902.5

29% Cushion

A	B	C	D	E	F
29% Cushion	V (µl)	Stock conc		Final conc
Optiprep	15225	60	%	29	%
ODN	16275
Total Volume	31500	4500	per sample

Rinse douncers with some HB buffer – leave these On ice too.

Add 1mL HB to douncer.

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. ~100mg of tissue works well.

Homogenise:20 strokes with pestle A and then80 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.

Keep douncer with homogenised sample On ice for and repeat steps 3-6 for other samples.

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

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

Add 4mL of optiprep cushion to an ultracentrifuge tube On ice for each sample.

Layer 5.25mL 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 200µL-400µL 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.

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.

Add samples to ultracentrifuge buckets.

Spin samples in SW41Ti 7700rpm,0h 0m 0s 0h 30m 0s 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.

Take out 10X beads from -80°C and GEM kit from-20°C. Prepare Template Switch Oligo as per 10x instructions if needed.

Prepare the resuspension buffer:

RNAseq Resuspension Buffer (for 6 samples + 1 for waste). Prepare and keep on ice.

A	B	C	D	E	F
RNAseq Resuspension Buffer
	V (uL)	stock		final
1x PBS ( w/o Ca and Mg)	9464
BSA	1680	10	%	1.5	%
RNase IN (Promega Purple cap)	56	40	u/uL	0.2	u/uL
final volume	11200

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 200µL - 500µL 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.

Add in 200µL 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.

Wash out ultracentrifuge tube with further 200µL of suspension buffer, pool in the LoBind tube On ice.

Add a further 400µL of suspension buffer to the LoBind tube On ice (800µL total suspension buffer).

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.

Keep On ice until the other samples have been resuspended and filtered.

Centrifuge 500rcf,0h 0m 0s for 0h 5m 0s and at 4°C.

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 400µL resuspension buffer by pipetting.

Filter through a filter-cap Falcon tube.

Transfer to LoBind Eppendorf On ice and centrifuge 500rcf,0h 0m 0s for 0h 5m 0s and at 4°C.

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

Filter through a filter-cap Falcon tube. Keep On ice.

Count each aliquot with Luna FL (18µL sample + 2µL Acridine Orange/Propidium Iodide). Add 10µL to slide. Save images and counts.

Create a new RNAseq aliquot of 1000 nuclei/uL in 80µL by diluting original sample.

Count diluted sample and record number (save files if needed but adds time for each sample).

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.