Fixed RNA - FFPE Resection Tissue (gentleMACS dissociation)

Buffer Preparation (~ min): 0h 20m 0s min):

Prepare Dissociation Enzyme Mix; incubate at 37°C for 0h 10m 0smin before proceeding with dissociation:

A	B	C	D	E	F
Dissociation Enzyme Mix	Stock	Final	1 rxn (µl)	4 rxn + 10% (µl)	8 rxn + 10% (µl)
Liberase TH (mg/ml)	5	1	420	1848	3696
RPMI	-	-	1680	7392	14,784
Total Volume (µl)			2100	9240	18,480

Reconstitute Liberase TH using 1mL Nuclease-free water.

Prepare Quenching Buffer, maintain at 4°C:

A	B	C	D	E	F
Quenching Buffer	Stock	Final	1 rxn (µl)	4 rxn + 10% (µl)	8 rxn + 10% (µl)
Nuclease-free water	-	-	437.5	1925	3850
Conc. Quench Buffer (10x Genomics)	8X	1X	62.5	275	550
Total Volume (µl)			500	2200	4400

Thaw Quench Buffer at room temperature, keep on ice.

Prepare fresh 70% and 50% Ethanol (1 ml each/sample).

Transfer either one 50 µm or two 25 µm FFPE scrolls to a gentleMACS C tube keeping the scrolls intact.

Note: Scrolls need to be intact and remain intact during the subsequent steps until the gentleMACS run. If scrolls appear as shards, request new scrolls from the biorepository. If scrolls disintegrate after adding xylene, take extra care when aspirating solutions.

Add 3mL Xylene; incubate for 0h 10m 0s min.

Remove without breaking the scrolls.

Repeat Step 3 and 3.1.

Add 3mL 100% Ethanol; incubate for 0h 0m 30s sec.

Remove without breaking the scrolls.

Repeat sequentially (0h 0m 30s sec each) with 1mL 100% Ethanol, 1mL 70% Ethanol, 1mL 50% Ethanol.

Add 1mL Nuclease-free water; incubate for 30 sec.

Remove without breaking the scrolls.

Add 1mL chilled PBS; maintain on ice.

When ready to begin gentleMACS Octo Dissociator, remove PBS.

Add 2mL Dissociation Enzyme Mix; secure cap, attach to gentleMACS with heater attached and run the following program (duration: ~0h 48m 0s min):

A	B
temp ON
spin - 20 rpm	5' 0"
loop 3X
spin 20 rpm	14' 0"
spin 1700 rpm	7"
spin 1700 rpm	1"
spin -1700 rpm	2"
spin 1700 rpm	1"
spin 1700 rpm	4"
end loop
end

saved as 'fixed_ffpe' program

Centrifuge 300rcf min.

Resuspend pellet in supernatant; filter through a 30µm strainer.

Wash strainer with 2mL chilled PBS (~4mL total volume).

Centrifuge 850rcf min.

Remove supernatant.

Add 500µL Tissue Resuspension Buffer; resuspend pellet.

Count cells using below protocol and keep in mind that concentrations provided by the counter assume 1mL volume, but the actual volume is half.

Counting Cells Using Cellaca MX

Confirm cells numbers are in the correct range to move forward with hybridization:

• If <100,000 total cells, do not move forward to hybridization. Consider having thicker scrolls cut and repeat the procedure.
• If >2,000,000 total cells, divide resuspension before centrifuging in step 16 (ideally, you would proceed with 1 - 2 million cells).

Reagent Preparation (~ min): 0h 20m 0s min):

Thaw Hyb Buffer B at 42°C. Vortex and centrifuge briefly. Keep warm and verify no precipitate before use.

DO NOT keep the thawed buffer on ice, or the solution will precipitate.

Thawed Hyb Buffer B can be kept at 42°C for up to 1h 0m 0s hour.

Thaw Enhancer for 0h 10m 0s min at 65°C. Vortex and centrifuge briefly. Keep warm and verify

no precipitate before use.

DO NOT keep the thawed reagent on ice, or the solution will precipitate.

Once thawed, Enhancer can be kept at 42°C for up to 0h 10m 0s min.

Thaw Human WTA Probes on ice. Vortex and centrifuge briefly.

Prepare Hyb Mix at Room temperature. Pipette mix 10x.

A	B	C	D	E	F
Hyb Mix (add reagents in order listed)	PN	1X (µl)	1X + 20% (µl)	4X + 20% (µl)	16X + 20% (µl)
Hyb Buffer B	2000485/ 2000483	70	84	336	1344
Enhancer	2000482	10	12	48	192
Total (µl)		80	96	384	1536

Ensure Enhancer has been incubated at 65C for 10 mins prior to use.

Incubate Hyb Mix at 42°C for 0h 5m 0s min.

Set thermal cycler to the following program:

A	B	C
Step	Temperature	Time
Pre-equilibriate	42C	Hold
Probe hybridization	42C	24 h*

Saved as 'hybridization'Lid Temperature: Reaction Volume: Runtime: Overnight *24 h is the maximum incubation, be mindful of this time depending on experiment.

Centrifuge fixed cells/nuclei resuspended in Quenching Buffer at 850rcf min at 4°C. 

Remove the supernatant.

Resuspend each pellet in 80µL prepared Hyb Mix (from step 15.4) and transfer to a pcr tube strip. Keep sample at room temperature. DO NOT place on ice.

Add 20µL unique single Human WTA Probes to the 80 μl mixture of Hyb Mix and fixed sample and gently pipette mix 10x with pipette set at 80 μl. Record the Human/Mouse WTA Probes name and part number used for each sample.

Incubate sample for 16h 0m 0s to 24h 0m 0s hours in preset thermal cycler program.

Incubation time should be consistent across all samples in an experiment.

Reagent Preparation (~ min): 0h 20m 0s min):

Thaw Enhancer for 0h 10m 0s min at 65°C. Vortex and centrifuge briefly. Keep warm and verify no precipitate before use. DO NOT keep the thawed reagent on ice, or the solution will precipitate.

Once thawed, Enhancer can be kept at 42°C for up to 0h 10m 0s min.

Thaw Conc. Post-Hyb Buffer at Room temperature and keep on ice.

Prepare Post-Hyb Wash Buffer. Vortex briefly and keep at Room temperature. DO NOT keep at 4C.

These volumes are sufficient for 1 well with 10% overage (4 or 16 samples). If loading more than 1 well, adjust volumes accordingly.

A	B	C	D
Post-Hyb Wash Buffer (add reagents in order listed)	PN	Pooling 4 samples (mL)	Pooling 16 samples (mL)
Nuclease-free water	-	4.95	13.86
Hyb Buffer B	2000533	0.275	0.77
Enhancer	2000482	0.275	0.77
Total (µl)		5.5	15.40

Note: volumes are in ml not µl.

Remove tubes from thermal cycler (8-tube strips) after overnight incubation.

Dilute each sample by adding 190µL of Post-Hyb Wash Buffer prepared in step 21 and pipette mix 5x.

Count cells in duplicate using below protocol and keep in mind that concentrations provided by the counter assume 1mL volume, but the actual volume is half.

Counting Cells Using Cellaca MX

Enter cell concentrations (cells/µl) and sample volume in the Chromium Fixed RNA Profiling for Multiplexed Samples - Pooling Workbook to determine the volume required to normalize cell concentrations:

CG000565_ChromiumFixedRNAProfiling_MultiplexedSamples_PoolingWorkbook_RevA_TEMPLATE.xlsx

Pool an equal number of cells from different hybridization reactions into a 5-ml (for 4 pooling samples) or 15-ml (for pooling 16 samples) centrifuge tube.

Add 2.3mL Post-Hyb Wash Buffer (if multiplexing 4 samples) or add 9.2mL Post-Hyb Wash Buffer (if multiplexing 16 samples). Mix by inverting 5x.

Centrifuge pooled samples at 850rcf at Room temperature.

Remove the supernatant without disturbing the pellet. Use a swinging bucket rotor if <500,000 cells.

Note: When performing post-hybridization washing with low cell numbers (i.e. <500,000 cells), complete removal of the supernatant is not required. Up to 30 µl of supernatant may be left behind to optimize cell recovery without significantly impacting assay performance.

Resuspend cell pellet in 1mL Post-Hyb Wash Buffer and transfer to a 1.5 mL microcentrifuge tube.

Incubate at 42°C for 0h 10m 0s min in a thermomixer or a heat block.

Centrifuge at 850rcf min at Room temperature.

Remove the supernatant without disturbing the pellet.

Resuspend cell pellet in 0.5mL Post-Hyb Wash Buffer . Pipette mix 5x.

Repeat steps in 29 - 29.4 three more times for a total of 4 washes using 0.5mL Post-Hyb Wash Buffer.

During the final centrifuge step in 29.5, prepare Post-Hyb Resuspension Buffer. Pipette mix 10x and maintain at 4°C:

A	B	C
Post-Hyb Resuspension Buffer (Add reagents in the order listed)	PN	1 Pool + 10% (µl)
Nuclease-free water	-	1567.5
Conc. Post-Hyb Buffer	2000533	82.5
Total		1650.0

Resuspend cell pellet in an appropriate volume of chilled Post-Hyb Resuspension Buffer. The buffer volume will depend upon the starting number of cells in the pool (table below). Pipette mix 20x to resuspend and breakup any cell clumps and maintain On ice.

A	B
Starting Total Cell Number in Pool	Post-Hyb Resuspension Buffer (µl)
<1 x 10^6	550
1 x 10^6 - 4 x 10^6	800
5 x 10^6 - 8 x 10^6	1050
9 x 10^6 - 12 x 10^6	1300
13 x 10^6 - 16 x 10^6	1550

Volumes reflect a 50µl overage to account for the subsequent counting step.

Pass the sample through a 30 μm filter (Sysmex CellTrics or Miltenyi Biotec Pre-Separation Filters) into a new 1.5-ml/2-ml microcentrifuge tube and place On ice.

Count cells in duplicate using below protocol and keep in mind that concentrations provided by the counter assume 1mL volume, but the actual volume is half.

If the sample concentration is not sufficient to achieve the desired targeted cell recovery, concentrate the sample as follows:

• Centrifuge a known volume of sample at 850 rcf for 5 min at room temperature.
• Carefully remove only a fraction of the supernatant, and pipette thoroughly to resuspend the cell pellet in the remaining volume. The amount of supernatant removed should be proportional to the desired increase in concentration.
• For example, to increase the concentration 4-fold from a starting volume of 400 μl, centrifuge, then remove 300 μl supernatant, and finally resuspend the cell pellet in the remaining 100 μl (400/100 = 4).
• Recount to confirm final concentration.

Reagent Preparation (~ min): 0h 30m 0s min):

Equilibrate Single Cell TL v1 Gel Beads to Room temperature 0h 30m 0s min before loading the chip.

Equilibriate Reducing Agent B to Room temperature. Vortex, verify no precipitate, centrifuge briefly.

Thaw GEM Reagent Mix at Room temperature. Vortex, verify no precipitate, centrifuge briefly. Keep On ice.

Keep GEM Enzyme Mix at -20°C until ready to use. Centrifuge briefly before

adding to the mix.

Prepare Master Mix On ice. Pipette mix 15x and centrifuge briefly.

A	B	C
GEM Master Mix (Add reagents in the order listed)	PN	1X* (µl)
GEM Reagent Mix	2000491	20.9
Reducing Agent B	2000087	1.7
GEM Enzyme Mix	2000490	12.4
Total		35.0

*1X = 1 well reaction. If loading more wells scale volumes accordingly.

Consult the attached tables to determine the correct ratio of sample to Post-Hyb Resuspension Buffer based on cell concentration and targeted cell recovery.

Cell Suspension Volume Calculator for Multiplexing 4 or 16 Samples.pdf

Add 35µL of prepared GEM Master Mix into each tube containing diluted sample and immediately proceed to the next step.

Assemble Chromium Next GEM Chip Q as follows:

1. Close the holder lid. Attach the gasket by holding the tongue (curved end, to the right) and hook the gasket on the left-hand tabs of the holder. Gently pull the gasket toward the right and hook it on the two right-hand tabs.
2. DO NOT touch the smooth side of the gasket.
3. Open the chip holder.
4. Remove the chip from the sealed bag. Use the chip within ≤ 24 h.
5. Align notch on the chip (upper left corner) and the open holder with the gasket attached.
6. Slide the chip to the left until the chip is inserted under the guide on the holder. Depress the right hand side of the chip until the spring-loaded clip engages.
7. Keep the assembled unit with the attached gasket open until ready for and while dispensing reagents into the wells.
8. DO NOT touch the smooth side of the gasket.
9. After loading reagents, close the chip holder. DO NOT press down on the top of the gasket.

Load Chromium Next GEM Chip Q as follows.

When loading the chip, raising and depressing the pipette plunger should take ~5 sec. When dispensing, raise the pipette tips at the same rate as the liquid is rising, keeping the tips slightly submerged.

Add 50% glycerol solution to each unused well.

• 70µL in each unused well in Row 1 .
• 50µL in each unused well in Row 2 .
• 45µL in each unused well in Row 3 .

DO NOT add 50% glycerol to the bottom row of unused wells.

Prepare Gel Beads:

• Snap the tube strip holder with the Gel Bead strip onto a 10x Vortex Adapter. Vortex 0h 0m 30s sec.
• Centrifuge Gel Bead strip for ~0h 0m 5s sec. Confirm there are no bubbles at the bottom of the tubes & the liquid levels are even.
• Place the Gel Bead strip back in the holder. Secure the holder lid.

Load Row 1:

• With the pipette set to 70µl, gently pipette the GEM Master Mix + Sample 15x.
• Using the same pipette tips, dispense 70µL GEM Master Mix + Sample into the bottom center of wells in Row 1 without introducing bubbles.

Load Row 2:

• Puncture the foil seal of the Gel Bead tubes. Slowly aspirate 50µLGel Beads.
• Dispense into the wells in Row 2 without introducing bubbles.
• Wait 0h 1m 0s min.

Load Row 3:

• Dispense 45µL Partitioning Oil into the wells in Row 3 from a reagent reservoir.

Close the lid (gasket already attached). DO NOT touch the smooth side of the gasket. DO NOT press down on the top of the gasket.

Run the Chromium iX:

• Press the eject button on the Chromium X to eject the tray. If the eject button is not touched within 1 min, tray will close automatically. System requires a few seconds before the tray can be ejected again.
• Place the assembled chip with the gasket in the tray, ensuring that the chip stays horizontal. Press the button to retract the tray.
• Press the play button.
• At completion of the run (~5.5 min), Chromium X/iX will chime.

Immediately proceed to the next step.

Transfer GEMs:

• Place a tube strip on ice.
• Press the eject button of the Chromium X/iX and remove the chip.
• Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees.
• Check the volume in Rows 1-2. Abnormally high volume in any well indicates a clog.
• Slowly aspirate 100µL GEMs from the lowest points of the recovery wells in Row 3 (top of chip) without creating a seal between the tips and the bottom of the wells.
• Withdraw pipette tips from the wells. GEMs should appear opaque and uniform across all channels.
• Over the course of ~0h 0m 20s sec, dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the tubes.

Incubate in a thermal cycler with the following protocol:

A	B	C
Step	Temperature	Time
1	25C	60 min
2	60C	45 min
3	80C	20 min
Hold	4C	Hold

Lid Temperature: 80CReaction Volume: 100µlRun Time: ~125 min

Store at for up to 1 week, or proceed to: 4°C for up to 1 week, or proceed to: