TITLE: Fixed RNA - FFPE Resection Tissue (gentleMACS dissociation)
AUTHORS: Ksenija Sabic

[DESCRIPTION]
This protocol utilizes the gentleMACS OctoDissociator, for pestle dissociation please refer to the original 10x protocol. This protocol assumes multiplexing exactly 4 or 16* samples with 8,000 cells per sample, if fewer samples are being multiplexed or sub-pools are required, refer to the the 10x protocol for pooling recommendations. 

*If 16 samples are being pooled, it is recommended to divide the samples into two batches due to the 8 sample limitation of the gentleMACS OctoDissociator.

For video instructions: https://www.10xgenomics.com/support/single-cell-gene-expression-flex

[BEFORE_START]
Set  water bath to  65 °C .
Set heat block to 42 °C .
Chill PBS in 4 °C if not already.


[GUIDELINES]
Please review and consult the full 10x Genomics protocols prior to starting and at any point during the procedure if needed.  

[STEPS]
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
1. 
Buffer Preparation (~20 min min):
 

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
2. Transfer either one   or two   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.

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
1.1. Prepare Dissociation Enzyme Mix; incubate at 37 °C for 10 minmin before proceeding with dissociation:
 

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
1.2. Prepare Quenching Buffer, maintain at 4 °C:

 

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
1.3. Prepare fresh 70% and 50% Ethanol (1 ml each/sample).
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
3. Add 3 mL Xylene; incubate for 10 min min.

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
3.1. Remove without breaking the scrolls.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
3.2. Repeat Step 3 and 3.1.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
4. Add 3 mL 100% Ethanol; incubate for 30 s sec.

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
4.1. Remove without breaking the scrolls.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
5. Repeat sequentially (30 s sec each) with 1 mL 100% Ethanol, 1 mL 70% Ethanol, 1 mL 50% Ethanol.

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
6. Add 1 mL Nuclease-free water; incubate for 30 sec.

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
6.1. Remove without breaking the scrolls.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
7. Add 1 mL chilled PBS; maintain on ice.

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
7.1. When ready to begin gentleMACS Octo Dissociator, remove PBS.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
8. Add 2 mL Dissociation Enzyme Mix; secure cap, attach to gentleMACS with heater attached and run the following program (duration: ~48 min min):
 

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
9. Centrifuge 300 rcf, 1 min min.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
10. Resuspend pellet in supernatant; filter through a 30µm strainer.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
10.1. Wash strainer with 2 mL chilled PBS (~4 mL total volume).

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
11. Centrifuge 850 rcf, 5 min min.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
11.1. Remove supernatant.
SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
12. Add 500 µL Tissue Resuspension Buffer; resuspend pellet.

SECTION: Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
13. Count cells using below protocol and keep in mind that concentrations provided by the counter assume 1mL volume, but the actual volume is half. 
 

14. 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).

SECTION: Probe Hybridization
15. Reagent Preparation (~ 20 min min):

SECTION: Post-Hybridization Pool &amp; Wash
20. Reagent Preparation (~20 min min): 
SECTION: Probe Hybridization
15.1. 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 60 min hour.
SECTION: Probe Hybridization
15.2. Thaw Enhancer for 10 min 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 10 min min.
SECTION: Probe Hybridization
15.3. Thaw Human WTA Probes on ice. Vortex and centrifuge briefly.
SECTION: Post-Hybridization Pool &amp; Wash
20.1. Thaw Enhancer for 10 min 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 10 min min.
SECTION: Post-Hybridization Pool &amp; Wash
20.2. Thaw Conc. Post-Hyb Buffer at Room temperature and keep on ice.

SECTION: Post-Hybridization Pool &amp; Wash
21. 
 
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.
 

SECTION: Post-Hybridization Pool &amp; Wash
22. Remove tubes from thermal cycler (8-tube strips) after overnight incubation.
SECTION: Post-Hybridization Pool &amp; Wash
23. Dilute each sample by adding 190 µL of Post-Hyb Wash Buffer prepared in step 21 and pipette mix 5x.
SECTION: Post-Hybridization Pool &amp; Wash
24. 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.  
 


SECTION: Post-Hybridization Pool &amp; Wash
25. 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:
  
SECTION: Post-Hybridization Pool &amp; Wash
26. 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.
SECTION: Post-Hybridization Pool &amp; Wash
27. Add 2.3 mL Post-Hyb Wash Buffer (if multiplexing 4 samples) or add 9.2 mL Post-Hyb Wash Buffer (if multiplexing 16 samples). Mix by inverting 5x.

SECTION: Post-Hybridization Pool &amp; Wash
28. Centrifuge pooled samples at 850 rcf, 5 min at Room temperature.

SECTION: Post-Hybridization Pool &amp; Wash
28.1. 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.
SECTION: Post-Hybridization Pool &amp; Wash
29. Resuspend cell pellet in 1 mL Post-Hyb Wash Buffer and transfer to a 1.5 mL microcentrifuge tube.

SECTION: Post-Hybridization Pool &amp; Wash
29.1. Incubate at 42 °C for 10 min min in a thermomixer or a heat block.

SECTION: Post-Hybridization Pool &amp; Wash
29.2. Centrifuge at 850 rcf, 5 min min at Room temperature.

SECTION: Post-Hybridization Pool &amp; Wash
29.3. Remove the supernatant without disturbing the pellet.
SECTION: Post-Hybridization Pool &amp; Wash
29.4. Resuspend cell pellet in 0.5 mL Post-Hyb Wash Buffer. Pipette mix 5x.

SECTION: Post-Hybridization Pool &amp; Wash
29.5. Repeat steps in 29 - 29.4 three more times for a total of 4 washes using 0.5 mL Post-Hyb Wash Buffer.
SECTION: Post-Hybridization Pool &amp; Wash
30. During the final centrifuge step in 29.5, prepare Post-Hyb Resuspension Buffer. Pipette mix 10x and maintain at 4 °C:
 


SECTION: Post-Hybridization Pool &amp; Wash
31. 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.
 


SECTION: Post-Hybridization Pool &amp; Wash
32. 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.
SECTION: Post-Hybridization Pool &amp; Wash
33. 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.  
SECTION: Post-Hybridization Pool &amp; Wash
33.1. 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.

SECTION: GEM Generation and Barcoding
34. Reagent Preparation (~ 30 min min):

SECTION: GEM Generation and Barcoding
34.1. Equilibrate Single Cell TL v1 Gel Beads to Room temperature 30 min min before loading the chip.

SECTION: GEM Generation and Barcoding
34.2. Equilibriate Reducing Agent B to Room temperature. Vortex, verify no precipitate, centrifuge briefly.
SECTION: GEM Generation and Barcoding
34.3. Thaw GEM Reagent Mix at Room temperature. Vortex, verify no precipitate, centrifuge briefly. Keep on ice.

SECTION: GEM Generation and Barcoding
34.4. Keep GEM Enzyme Mix at -20 °C until ready to use. Centrifuge briefly before
adding to the mix.

SECTION: GEM Generation and Barcoding
35. Prepare Master Mix on ice. Pipette mix 15x and centrifuge briefly.
 

SECTION: Probe Hybridization
15.4. Prepare Hyb Mix at Room temperature. Pipette mix 10x. 
 

SECTION: Probe Hybridization
15.5. Incubate Hyb Mix at 42 °C for 5 min min.
SECTION: Probe Hybridization
15.6. Set thermal cycler to the following program:
 
*24 h is the maximum incubation, be mindful of this time depending on experiment.
SECTION: Probe Hybridization
16. Centrifuge fixed cells/nuclei resuspended in Quenching Buffer at 850 rcf, 5 min min at 4 °C. 
SECTION: Probe Hybridization
17. Resuspend each pellet in 80 µL prepared Hyb Mix (from step 15.4) and transfer to a pcr tube strip. Keep sample at room temperature. DO NOT place on ice.
SECTION: Probe Hybridization
16.1. Remove the supernatant.
SECTION: Probe Hybridization
18. Add 20 µL 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.
SECTION: Probe Hybridization
19. Incubate sample for 960 min to 1440 min hours in preset thermal cycler program.

Incubation time should be consistent across all samples in an experiment.
SECTION: GEM Generation and Barcoding
36. Consult the attached tables to determine the correct ratio of sample to Post-Hyb Resuspension Buffer based on cell concentration and targeted cell recovery.

  

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

SECTION: GEM Generation and Barcoding
38. Assemble Chromium Next GEM Chip Q as follows: 
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.
DO NOT touch the smooth side of the gasket.
Open the chip holder.
Remove the chip from the sealed bag. Use the chip within ≤ 24 h.
Align notch on the chip (upper left corner) and the open holder with the gasket attached.
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.
Keep the assembled unit with the attached gasket open until ready for and while dispensing reagents into the wells.
DO NOT touch the smooth side of the gasket.
After loading reagents, close the chip holder. DO NOT press down on the top of the gasket.
SECTION: GEM Generation and Barcoding
39. 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.
SECTION: GEM Generation and Barcoding
39.1. Add 50% glycerol solution to each unused well. 
70 µL in each unused well in Row 1.
50 µL in each unused well in Row 2.
45 µL in each unused well in Row 3.

DO NOT add 50% glycerol to the bottom row of unused wells.
SECTION: GEM Generation and Barcoding
39.2. Prepare Gel Beads:
Snap the tube strip holder with the Gel Bead strip onto a 10x Vortex Adapter. Vortex 30 s sec.
Centrifuge Gel Bead strip for ~5 s 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.

SECTION: GEM Generation and Barcoding
39.3. Load Row 1:
With the pipette set to 70µl, gently pipette the GEM Master Mix + Sample 15x.
Using the same pipette tips, dispense 70 µL GEM Master Mix + Sample into the bottom center of wells in Row 1 without introducing bubbles.
SECTION: GEM Generation and Barcoding
39.4. Load Row 2:
Puncture the foil seal of the Gel Bead tubes. Slowly aspirate 50 µLGel Beads.
Dispense into the wells in Row 2 without introducing bubbles.
Wait 1 min min.
SECTION: GEM Generation and Barcoding
39.5. Load Row 3:
Dispense 45 µL Partitioning Oil into the wells in Row 3 from a reagent reservoir.

SECTION: GEM Generation and Barcoding
39.6. 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.
SECTION: GEM Generation and Barcoding
40. 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.
SECTION: GEM Generation and Barcoding
41. 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 100 µL 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 ~20 s sec, dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the tubes.
SECTION: GEM Generation and Barcoding
42. Incubate in a thermal cycler with the following protocol:
 

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