Dec 21, 2023

Fixed RNA - FFPE Resection Tissue (gentleMACS dissociation) V.1

DOI
https://dx.doi.org/10.17504/protocols.io.eq2lyj4wrlx9/v1
Fixed RNA - FFPE Resection Tissue (gentleMACS dissociation)
  • 1Icahn School of Medicine at Mount Sinai
Ksenija Sabic
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2lyj4wrlx9/v1
Protocol Citation: Ksenija Sabic 2023. Fixed RNA - FFPE Resection Tissue (gentleMACS dissociation). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyj4wrlx9/v1
Manuscript citation:



License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: December 15, 2023
Last Modified: January 17, 2024
Protocol  Integer ID: 92347
Keywords: ffpe resection tissue, fixed rna, cells per sample, single cell, gentlemacs dissociation, sample limitation of the gentlemacs octodissociator, gentlemacs octodissociator, rna, cell, 10x protocol, original 10x protocol
Abstract
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
Image Attribution
10x Genomics
Guidelines
Please review and consult the full 10x Genomics protocols prior to starting and at any point during the procedure if needed.
Materials
From 10x Genomics:
  • Conc. Quench Buffer10x GenomicsCatalog #2000516
Note
**Included in the 10x Genomics Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit, 16 rxns (PN-1000414); also includes Enhancer (PN-2000482) if storing fixed cells.
  • Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 4 BC10x GenomicsCatalog #1000475
OR
  • Chromium Fixed RNA Kit, Human Transcriptome, 4 rxns x 16 BC10x GenomicsCatalog #1000476
  • Chromium Next GEM Chip Q Single Cell Kit, 48 rxns10x GenomicsCatalog #1000418
OR
  • Chromium Next GEM Chip Q Single Cell Kit, 16 rxns10x GenomicsCatalog #1000422
  • Dual Index Kit TS Set A, 96 rxns10x GenomicsCatalog #1000251

Miscellaneous:
  • XyleneMerck MilliporeSigma (Sigma-Aldrich)Catalog #214736
Safety information
Xylene is a highly flammable liquid and vapor causes irritation to eyes, skin, and respiratory tract. Only use in chemical hood and dispose in designated Xylene waste container.

  • Liberase THMerck MilliporeSigma (Sigma-Aldrich)Catalog #5401135001
  • GentleMACS C tubeMiltenyi BiotecCatalog #130-093-237
  • VitaStain AOPI Staining SolutionNexcelomCatalog #CS2-0106-5ml
  • Cell Counting Plates
  • Pre-Separation Filters (30 µm)Miltenyi BiotecCatalog #130-041-407
  • PBS 1x without calcium & magnesiumVWR International (Avantor)Catalog #Cat# 21-040-CVR or similar
  • RPMI 1640 with L-glutamineCorningCatalog #10040CV or similar
  • Ethyl alcohol, 200 proof, anhydrous, ≥99.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #459836 or similar
  • Nuclease-Free Water (not DEPC-Treated)Thermo Fisher ScientificCatalog #AM9937 or similar


Equipment
gentleMACS™ Dissociator
NAME
tissue dissociator
TYPE
Miltenyi Biotec
BRAND
130-093-235
SKU
https://www.miltenyibiotec.com/US-en/products/gentlemacs-dissociator.html
LINK
8 tubes, etc..
SPECIFICATIONS

Equipment
Cellaca MX High-throughput Automated Cell Counter
NAME
cell counter
TYPE
Nexcelom
BRAND
MX0112-0127
SKU




Safety warnings
Perform Xylene steps in chemical hood. During the first Ethanol incubation, samples may be moved to the bench.
Before start
Set water bath to 65 °C .
Set heat block to 42 °C .
Chill PBS in 4 °C if not already.

Isolation of Cells from FFPE Tissue Sections for Chromium Fixed RNA Profiling
10m

Buffer Preparation (~00:20:00 min):
Note
All buffers should be prepared fresh.

20m
Prepare Dissociation Enzyme Mix; incubate at 37 °C for 00:10:00 min before proceeding with dissociation:
ABCDEF
Dissociation Enzyme MixStockFinal1 rxn (µl)4 rxn + 10% (µl)8 rxn + 10% (µl)
Liberase TH (mg/ml)5142018483696
RPMI--1680739214,784
Total Volume (µl)2100924018,480
Reconstitute Liberase TH using 1mL Nuclease-free water.

10m
Prepare Quenching Buffer, maintain at 4 °C :

ABCDEF
Quenching BufferStockFinal1 rxn (µl)4 rxn + 10% (µl)8 rxn + 10% (µl)
Nuclease-free water--437.519253850
Conc. Quench Buffer (10x Genomics)8X1X62.5275550
Total Volume (µl)50022004400
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 3 mL Xylene; incubate for 00:10:00 min.

10m
Remove without breaking the scrolls.
Repeat Step 3 and 3.1.
Add 3 mL 100% Ethanol; incubate for 00:00:30 sec.

30s
Remove without breaking the scrolls.
Repeat sequentially (00:00:30 sec each) with 1 mL 100% Ethanol, 1 mL 70% Ethanol, 1 mL 50% Ethanol.

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

Remove without breaking the scrolls.
Add 1 mL chilled PBS; maintain on ice.

When ready to begin gentleMACS Octo Dissociator, remove PBS.
Add 2 mL Dissociation Enzyme Mix; secure cap, attach to gentleMACS with heater attached and run the following program (duration: ~00:48:00 min):
AB
temp ON
spin - 20 rpm5' 0"
loop 3X
spin 20 rpm14' 0"
spin 1700 rpm7"
spin 1700 rpm1"
spin -1700 rpm2"
spin 1700 rpm1"
spin 1700 rpm4"
end loop
end
saved as 'fixed_ffpe' program

48m
Centrifuge 300 rcf, 00:01:00 min.
1m
Resuspend pellet in supernatant; filter through a 30µm strainer.
Wash strainer with 2 mL chilled PBS (~4 mL total volume).

Centrifuge 850 rcf, 00:05:00 min.
5m
Remove supernatant.
Add 500 µL 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.
Protocol
NAME
Counting Cells Using Cellaca MX
CREATED BY
Ksenija Sabic

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).

Probe Hybridization
1h 50m
Reagent Preparation (~ 00:20:00 min):

20m
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 01:00:00 hour.
1h
Thaw Enhancer for 00:10:00 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 00:10:00 min.
20m
Thaw Human WTA Probes on ice. Vortex and centrifuge briefly.
Prepare Hyb Mix at Room temperature . Pipette mix 10x.
ABCDEF
Hyb Mix (add reagents in order listed)PN1X (µl)1X + 20% (µl)4X + 20% (µl)16X + 20% (µl)
Hyb Buffer B2000485/ 200048370843361344
Enhancer2000482101248192
Total (µl)80963841536
Ensure Enhancer has been incubated at 65C for 10 mins prior to use.

Incubate Hyb Mix at 42 °C for 00:05:00 min.
5m
Set thermal cycler to the following program:
ABC
StepTemperatureTime
Pre-equilibriate42CHold
Probe hybridization42C24 h*
Saved as 'hybridization'
Lid Temperature: 42 °C
Reaction Volume: 100 µL
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 850 rcf, 00:05:00 min at 4 °C . 
5m
Remove the supernatant.
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.
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.
Incubate sample for 16:00:00 to 24:00:00 hours in preset thermal cycler program.

Incubation time should be consistent across all samples in an experiment.
1d 16h
Post-Hybridization Pool & Wash
40m
Reagent Preparation (~00:20:00 min):
20m
Thaw Enhancer for 00:10:00 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 00:10:00 min.
20m
Thaw Conc. Post-Hyb Buffer at Room temperature and keep on ice.


Note
This protocol utilizes the Pooled Wash Workflow, for the Individual Wash Workflow consult the original 10x protocol.
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.
ABCD
Post-Hyb Wash Buffer (add reagents in order listed)PNPooling 4 samples (mL)Pooling 16 samples (mL)
Nuclease-free water-4.9513.86
Hyb Buffer B20005330.2750.77
Enhancer20004820.2750.77
Total (µl)5.515.40
Note: volumes are in ml not µl.

Remove tubes from thermal cycler (8-tube strips) after overnight incubation.
Dilute each sample by adding 190 µL 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.
Protocol
NAME
Counting Cells Using Cellaca MX
CREATED BY
Ksenija Sabic


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:
Download CG000565_ChromiumFixedRNAProfiling_MultiplexedSamples_PoolingWorkbook_RevA_TEMPLATE.xlsxCG000565_ChromiumFixedRNAProfiling_MultiplexedSamples_PoolingWorkbook_RevA_TEMPLATE.xlsx1.4MB
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 2.3 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.

Centrifuge pooled samples at 850 rcf, 00:05:00 at Room temperature .

5m
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 1 mL Post-Hyb Wash Buffer and transfer to a 1.5 mL microcentrifuge tube.

Incubate at 42 °C for 00:10:00 min in a thermomixer or a heat block.

10m
Centrifuge at 850 rcf, 00:05:00 min at Room temperature .

5m
Remove the supernatant without disturbing the pellet.
Resuspend cell pellet in 0.5 mL Post-Hyb Wash Buffer. Pipette mix 5x.

Repeat steps in 29 - 29.4 three more times for a total of 4 washes using 0.5 mL 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 :
ABC
Post-Hyb Resuspension Buffer (Add reagents in the order listed)PN1 Pool + 10% (µl)
Nuclease-free water-1567.5
Conc. Post-Hyb Buffer200053382.5
Total1650.0


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 .
AB
Starting Total Cell Number in PoolPost-Hyb Resuspension Buffer (µl)
<1 x 10^6550
1 x 10^6 - 4 x 10^6800
5 x 10^6 - 8 x 10^61050
9 x 10^6 - 12 x 10^61300
13 x 10^6 - 16 x 10^61550
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.

GEM Generation and Barcoding
1h
Reagent Preparation (~ 00:30:00 min):

30m
Equilibrate Single Cell TL v1 Gel Beads to Room temperature 00:30:00 min before loading the chip.

30m
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.
ABC
GEM Master Mix (Add reagents in the order listed)PN1X* (µl)
GEM Reagent Mix200049120.9
Reducing Agent B20000871.7
GEM Enzyme Mix200049012.4
Total35.0
*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.

Download Cell Suspension Volume Calculator for Multiplexing 4 or 16 Samples.pdfCell Suspension Volume Calculator for Multiplexing 4 or 16 Samples.pdf3.7MB

Add 35 µL 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.
  • 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.
Prepare Gel Beads:
  • Snap the tube strip holder with the Gel Bead strip onto a 10x Vortex Adapter. Vortex 00:00:30 sec.
  • Centrifuge Gel Bead strip for ~00:00:05 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.

35s
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.
Load Row 2:
  • Puncture the foil seal of the Gel Bead tubes. Slowly aspirate 50 µL Gel Beads.
  • Dispense into the wells in Row 2 without introducing bubbles.
  • Wait 00:01:00 min.
1m
Load Row 3:
  • Dispense 45 µL 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 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 ~00:00:20 sec, dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the tubes.
20s
Incubate in a thermal cycler with the following protocol:
ABC
StepTemperatureTime
125C60 min
260C45 min
380C20 min
Hold4CHold
Lid Temperature: 80C
Reaction Volume: 100µl
Run Time: ~125 min

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

Protocol references
10x Genomics Protocols:
CG000632 | Rev A
CG000527 | Rev E