CG000553 | Rev B

DEMONSTRATED PROTOCOL

Tissue Fixation & Dissociation
for Chromium Fixed RNA Profiling
Introduction
This protocol outlines how to perform tissue
fixation followed by dissociation of the fixed tissue
for use with Chromium Fixed RNA Profilingworkflow. Storage recommendations for the fixedtissue and post-storage processing are listed in the
Appendix.

Additional GuidanceThis protocol was demonstrated using 25 mg ofvarious mouse and human tissue types (flashfrozen and fresh), including brain, heart, liver,spleen, testes, ileum, duodenum, pancreas, kidney,lung, colon, tonsil, lymph, etc. See Appendix for acomplete list of tissues tested along withcorresponding cell yields. Optimize the protocol
based on tissue type, tissue density, anddissociation efficiency.
Tissue and cells carry potentially hazardous
pathogens. Follow material supplier recommendations and local laboratory procedures
and regulations for the safe handling, storage
and disposal of biological materials.

Specific Reagents & Consumables
VendorItem

For Tissue Mincing & Fixation
10x
Chromium Next GEM Single
Genomics
Cell Fixed RNA Sample
Preparation Kit
MilliporeProtector RNase Inhibitor
Sigma
Albumin, Bovine Serum, 10%
Aqueous Solution, NucleaseFree
ThermoUltraPure Bovine Serum
FisherAlbumin (BSA, 50 mg/ml)
Scientific
Alternative to Millipore Sigma
product
Formaldehyde (37% by
Weight/Molecular Biology),
Fisher BioReagents
Nuclease-free Water(not DEPC-Treated)
Pyrex Petri Dish*Part Number
10004143335399001
126615

AM2616

BP531-25

AM993708-748D

General-Purpose Forceps*

10-270Standard Dissecting Scissors*

08-951-20

Wide-Bore Pipette Tips RT
FLW 768A/8
LTS 1000 uL*(30389218)*May be procured from alternative vendors
Corning
VWR

Phosphate-Buffered Saline,
21-040-CV
1X without Calcium andMagnesium
Tris Buffer, 1M sterile solution, E199-100ML
pH 8.0Additional Materials
Blade; Lab Weighing Scale

This list may not include some standard laboratory equipments.

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CG000553 | Rev B

Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling
For Tissue Dissociation
Liberase TL
Millipore
Liberase TH (optional)
Sigma
Miltenyi
gentleMACS Octo Dissociator
Biotecwith Heaters
gentleMACS C Tubes
CorningCorning RPMI 1640

Preparation - Buffers
5401020001
5401135001
130-096-427

All buffer preparations should be fresh.
Buffers for Fixation - Prepare fresh (1 ml)
Fixation Buffer

Stock

Final

Nuclease-free Water--

Per 25 mg
tissue (μl)
791.9Conc. Fix & Perm Buffer*(10x Genomics PN 2000517)
Formaldehyde

10X

1X

100.0

37%

4%

108.1

Tissue Resuspension Buffer Stock
Maintain at 4oC

Final

Per sample
(μl)

PBS

1X

0.496X 496.0

Tris buffer (pH 8.0; mM)

1000

50

Miltenyi
MACS SmartStrainers (30 µm) 130-098-458
BiotecChoose either Sysmex or Miltenyi Biotec filter.

BSA (RNase free)

10%0.02% 2.0

RNase Inhibitor (U/μl)

40

0.24

6.0

Nuclease-free Water-

For Cell Counting

Quenching Buffer

Stock

Final

Nuclease-free Water

-

-

446.0
Per 25 mg
tissue (μl)
875.0Cellaca MX High-throughput† MX-112-0127
Automated Cell Counter

Conc. Quench Buffer*(10x Genomics PN 2000516)

8X

1X

125.0

Countess II FL Automated
Cell Counter†

Dissociation Solution Prepare 2 ml

ThermoGibco RPMI 1640
FisherScientific
Choose either Corning or Gibco RPMI

Maintain at room temperature

130-093-237
10-040-CV
11875093

For Sample Filtration
SysmexSterile Single-Pack CellTrics
Filters (30 µm)

Nexcelom
ViaStain AOPI Staining
Biosciences Solution

ThermoFisherScientific04-004-2326

CS2-0106-5mL

AMAQAF1000

Countess II FL AutomatedC10228Cell Counting Chamber Slides
Ethidium Homodimer-1

E1169†Choose either Countess or Cellaca.

Millipore
Sigma

VWR

Glycerol, 99.5%, for molecular 327255000
biology, DNAse, RNAse andProtease free
Alternative to Millipore Sigma
product
Glycerol for molecular
G5516-100ML
biology, ≥99.0% Alternative to
Acros Organics productProtector RNase Inhibitor

3335402001

Vacuum Filter/Storage
System (0.2 µm)

29442-936

Additional Materials
Eppendorf• Reconstitute 5mg Liberase by adding 1 ml sterile water.
Agitate at 2-8 °C until dissolved. Store stock solution insingle-use aliquots at -15 to -25°C• Prepare RPMI + 0.2 mg/ml Liberase (Add 80 µl Liberase
stock solution into 1,920 µl of RPMI, mix, maintain at 4oC)
• Warm Dissociation Solution for 10 min at 37oC before use.

For Storage & Post-Storage Processing
Acros
Organics

Maintain at 4oC

50.0

DNA LoBind Tubes 2.0 ml

022431048

ThermoMixer C

5382000023

This list may not include some standard laboratory equipments.

Additional Buffers
PBS + 0.04% BSA (maintain at 4oC)
Buffers for Storage of Fixed Samples - Prepare fresh
50% Glycerol Solution(needed for long-term storage of fixed samples)• Mix an equal volume of water and 99% Glycerol, Molecular
Biology Grade• Filter through a 0.2 μm filter• Store at room temperature in 2-ml LoBind tubes
Additional Buffers
0.5X PBS + 0.02% BSA (maintain at 4oC)
For post-storage processing; use RNase-free BSA*Included in the 10x Genomics Chromium Next GEM
Single Cell Fixed RNA Sample Preparation Kit, 16 rxns
(PN-1000414).

Formaldehyde should always be used with adequate ventilation, preferably in a fume hood. Follow appropriate regulations.
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2

Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

CG000553 | Rev B

Tips & Best Practices
The following recommendations are critical for optimal performance of the Chromium Fixed RNA
Profiling assay.

Tissue Quality & Processing

Fixed Cell Counting• Perform pilot experiments to determine if thetissue type is suitable for fixation and dissociation for preparing single cell suspension(s).

• Samples should have minimal debris for best
results; debris can have associated RNA that cancontribute to non-cell background.

• Ensure that variation in cell yields from different
tissue types is considered when performing theprotocol.

• Accurate sample counting is critical for optimal
assay performance.

• The tissue should be minced only on a glass
surface. DO NOT use plastic petri dish for
mincing tissue.

Centrifugation & Pellet Resuspension• It is strongly recommended that the sample be
stained with a fluorescent nucleic acid dye suchas Ethidium Homodimer-1 or AO/PI Staining
Solution and counted using an automated fluorescent cell counter (Countess II Automated Cell
Counter or a Cellaca counter).

• Use a swinging-bucket rotor for higher cell/nuclei
recovery.

• See Appendix for details on fixed cell counting.

• Centrifugation speed and time may need optimization depending upon the sample type.

Fixed Sample Storage• When working with samples with low cellnumbers (i.e. <500,000 cells), complete removalof the supernatant is not required. Up to 30 µl
supernatant may be left behind to optimize cellrecovery without significantly impacting assay
performance.

• Fixed tissue pieces and cell suspension derived
from fixed dissociated tissues can be stored at
4oC for up to 1 week or at -80oC for up to 6 months
after resuspending in appropriate reagents.
• Sample storage guidelines are provided in the
Appendix.

• After each buffer addition step, gently mix cells/
nuclei 5x, or until the pellet is completely resuspended, without introducing bubbles.

Up to 30 µl of
supernatant canbe left behind

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3CG000553 | Rev B

Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

Protocol Overview : Tissue Fixation & Dissociation
1. Tissue Fixation

Transfer to a
glass petri dish
Weigh the tissue

Mince tissue into
small pieces

Add Fixation Buffer(1 ml/25 mg tissue);pass through a widebore pipette tip

Incubate for
16-24 h at 4°C

850 rcf
5 min

Use scissors to cut any
remaining large pieces

Removesupernatant

Add Tissue Resuspension
or Quenching Buffer (1 ml);resuspend
tissue pellet

850 rcf
5 min

May be stored at 4oC
for up to 1 week/at -80oC for up to 6 months

Proceed to Fixed Tissue
Dissociation protocol

850 rcf
5 min

Add PBS (2 ml);resuspend
tissue pellet

RemovesupernatantRemovesupernatant

2. Fixed Tissue Dissociation

Transfer to a
gentleMACS C tube

May be dissociated manually(refer to protocol steps)

Attach the tube to
Octo Dissociator; run
appropriate program

Add Dissociation
Solution to fixedtissue pellet

Proceed to Chromium Fixed RNA
Profiling protocols(see References)

Filter through a
30 µm strainer;
wash filter with
PBS (2 ml)

Count fixed cells

850 rcf
5 min

Removesupernatant

Add Quenching Buffer (1 ml);resuspend pellet

May be stored at 4oC for up to 1 week/at -80oC for up to 6 months

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4Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

CG000553 | Rev B

Tissue Fixation & Dissociation Protocol
This protocol was demonstrated using 25 mg fresh or flash frozen tissue. For tissues with high cell density,less than 25 mg may also yield sufficient cell numbers. See Appendix for a complete list of tissues tested
along with corresponding cell yields from 25 mg of tissue. Chromium Next GEM RNA Profiling Sample
Fixation Kit, 16 rxns (PN-1000414) was used for tissue fixation.

1. Fix Tissue
If using frozen tissue, maintain the tissue on dry ice
before and after weighing. Use a pre-chilled petri
dish placed on ice while mincing the tissue.
a. Weigh tissue to determine Fixation Buffer
volume. 1 ml Fixation Buffer/25 mg tissue will
be used at step 1c.

g. Add 2 ml chilled PBS and resuspend the tissuepellet.
h. Centrifuge at 850 rcf for 5 min at room
temperature.
i.

Remove the supernatant without disturbing thetissue pellet.

j.

Add 1 ml Tissue Resuspension Buffer or
Quenching Buffer, resuspend the tissue pellet,
and maintain on ice.

b. Place the tissue on a pre-chilled glass petri dishmaintained on ice and using a blade, mince
tissue finely (enables passing through a 1 ml
wide-bore pipette tip).
c. Using a wide-bore (1.5 mm) 1-ml pipette, add
1 ml Fixation Buffer/25 mg tissue (see tablebelow). Pass the tissue up and down. Aspirate
the solution containing the minced tissue andtransfer to a 2 ml or larger centrifuge tube. Pass
the tissue up and down. If larger tissue pieces
fail to pass through the pipette, use dissection
scissors to cut the tissue pieces further until
they pass through the pipette. Maintain on ice.
Tissue Mass (mg)Fixation Buffer (ml)12-37 mg

1 ml

38-62 mg

2 ml

63-87 mg

3 ml

88-112 mg

4 ml

Fixed tissue pieces can be stored at 4oC for up to 1
week or at -80oC for up to 6 months. See Appendix
for guidance on short-term storage and post-storage
processing of fixed tissue pieces.
k. Centrifuge at 850 rcf for 5 min at room
temperature.
l.

Remove the supernatant without disturbing the
fixed tissue pellet.

m. Proceed to tissue dissociation.

d. Incubate for 16-24 h at 4°C.

DO NOT agitate or mix the sample during incubation.
Fixation time and temperature should be consistentacross all samples in an experiment.
e. Centrifuge at 850 rcf for 5 min at room
temperature.
f.

Remove the supernatant without disturbing thetissue pellet.
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5CG000553 | Rev B

Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

If tissue is tough to triturate and/or solution is
not cloudy after trituration, see Troubleshooting
guidance

2. Dissociate Fixed Tissue
a. Warm Dissociation Solution for 10 min at 37oC
before use.
b. Add 2 ml pre-warmed Dissociation Solution to
the sample.

d. Pass the dissociated tissue through a 30 µm filter
to remove debris and undissociated tissue pieces.

Use 2 ml Dissociation Solution for up to 100 mg
tissue. Use only 2 ml Dissociation Solution per
C tube.

e. Perform an additional wash of the 30 µm filterby adding 2 ml PBS to the filter. Collect thefiltrate in the same tube as step 2d.

c. Dissociate tissue. Using an Octo Dissociator ishighly recommended. Alternatively, tissue may
be dissociated manually.
• Octo Dissociator:
Transfer to Miltenyi C tubes. Use multiple
Miltenyi C tubes if tissue exceeds 100 mg and use
2 ml Dissociation Solution/C tube. Ensure thatno fixed tissue pieces are stuck on the walls of
the C tube. Place the C tube in the Octo Dissociator, apply Heating Units, and run the following
program:Use of swinging-bucket rotor is recommended forhigher cell recovery.
f.

g. Remove the supernatant without disturbing thepellet.
h. Resuspend pellet in 1 ml chilled Quenchingbuffer.
i.

gentleMACS Program
1
2
3
4
5

If the cell suspension contains aggregatesor debris larger than 30 µm, pass through a
30 µm filter and recount.

Dissociation can be monitored by taking a 10 µl
aliquot for counting. If the cell concentration islower than expected, additional dissociation may
be required. See Troubleshooting guidance.

OR• Manual Dissociation:
Incubate for 20 min at 37°C shaking the tube
intermittently. Using a P1000 pipette tip ora silanized glass pipette, triturate the tissue
pieces 15-20X (until solution begins to turncloudy) to obtain a single cell suspension.
Proceed to step 2d.

Determine the cell concentration using a Countess
II FL Automated Cell Counter or hemocytometer.
See Appendix for Fixed Cell Counting.

For accurate cell counting, it is strongly recommended that the cell/nuclei suspension be
stained with a fluorescent nucleic acid dye such as
Ethidium Homodimer-1 or AO/PI Staining Solution
and counted using an automated fluorescent cellcounter.

temp ON
spin 50 rpm, 20' 0"spin 1000 rpm, 30"
spin -1000 rpm, 30"
end• Detach the C tubes and proceed to step 2d.
OPTIONAL: Centrifuge the C tubes at 300 rcf
briefly to collect all the cells at the bottom of
the tube. Resuspend the pellet in the supernatant and proceed to step 2d.

Centrifuge at 850 rcf for 5 min.

For lower than expected cell yields, see
Troubleshooting guidance.
j.

Proceed immediately to appropriate
Chromium Fixed RNA Profiling protocols(see References) or store the sample after
resuspending in appropriate reagents.

Samples can be stored at 4oC for up to 1 week or
at -80oC for up to 6 months, depending upon thebuffers used. See Appendix for guidance on shortand long-term storage and post-storage processing of fixed tissue pieces and cells.

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6Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

CG000553 | Rev B

Appendix
Fixed Sample Storage Guidance

Fixed Tissue Pieces – Post-Storage Processing

Fixed samples can be stored for short or long-term.

Samples may undergo a color change duringstorage (e.g. black, light gray, or green), however
this will not impact assay performance.

Short-term Storage at 4oC(for fixed tissue pieces & cells)a. Thaw Enhancer for 10 min at 65°C. Vortex andcentrifuge briefly. Keep warm and verify no
precipitate before use.

DO NOT keep the thawed reagent on ice, or thesolution will precipitate. Once thawed, Enhancer
can be kept at 42°C for up to 10 min.
b. Add 0.1 volume pre-warmed Enhancer to fixedtissue pieces or cells in Tissue Resuspension
Buffer or fixed cells in Quenching Buffer. For
example, add 100 μl Enhancer to 1,000 μl fixed
cells in Quenching Buffer. Pipette mix.

If samples were stored at −80°C, thaw at room
temperature until no ice is present.
a. Centrifuge sample at 850 rcf for 5 min at room
temperature.
b. Remove the supernatant without disturbing thetissue pellet.
c. Proceed to Dissociate Fixed Tissue protocol(step 2a).

Fixed Cells – Post-Storage Processingc. Store sample at 4°C for up to 1 week.

Samples may undergo a color change duringstorage (e.g. black, light gray, or green), however
this will not impact assay performance.

Long-term Storage at −80°C(for fixed tissue pieces & cells)

If samples were stored at −80°C, thaw at room temperature until no ice is present.

a. Thaw Enhancer for 10 min at 65°C. Vortex andcentrifuge briefly. Keep warm and verify no
precipitate before use.

a. Centrifuge sample at 850 rcf for 5 min at room
temperature.

DO NOT keep the thawed reagent on ice, or thesolution will precipitate. Once thawed, Enhancer
can be kept at 42°C for up to 10 min.
b. Add 0.1 volume of pre-warmed Enhancer to
fixed sample in Quenching Buffer. For example,
add 100 μl Enhancer to 1,000 μl fixed sample in
Quenching Buffer. Pipette mix.
c. Add 50% Glycerol for a final concentration of
10%. For example: add 275 μl 50% Glycerol to
1,100 μl fixed sample in Quenching Buffer andEnhancer. Pipette mix.
d. Store at −80°C for up to 6 months.

Storing fixed cells at −80oC is recommended forbest results.

b. Remove the supernatant without disturbing thepellet.
c. Resuspend cell pellet in 1 ml Quenchinng
Buffer or 0.5X PBS + 0.02% BSA (optionallysupplemented with 0.2 U/μl RNase Inhibitor)and maintain on ice.

Use RNase-free BSA at this step. See Specific
Reagents & Consumables for details.
d. Determine cell concentration of the fixedsample using an Automated Cell Counter(Countess II/Cellaca MX) or hemocytometer.
See Appendix for Fixed Cell Counting.
e. Proceed immediately to appropriate
Chromium Fixed RNA Profiling protocols (seeReferences).

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7Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

Fixed Sample Shipping Guidancea. Fixed single cells resuspended in Quenching
Buffer or fixed tissue pieces resuspended in
Tissue Resuspension Buffer and supplemented
with Enhancer can be shipped with a cold pack.
See Short-term Storage for details.
b. Fixed single cells or fixed tissue pieces
resuspended in Quenching Buffer
supplemented with Enhancer and Glycerol canbe shipped on dry ice. See Long-term Storage
for details.

CG000553 | Rev B

Representative Cells Stained with Ethidium Homodimer-1

Optimize focus and exposure settings.

Fixed Cell/Nuclei Counting• Accurate sample counting is critical for optimal
assay performance.
• It is strongly recommended that the fixed sample
be stained with a fluorescent nucleic acid dyesuch as Ethidium Homodimer-1 or AO/PI staining
solution and counted using an automated fluorescent cell counter (Countess II Automated Cell
Counter or a Cellaca counter).

Counting Using Ethidium Homodimer-1
This protocol provides instructions for counting
samples using Ethidium Homodimer-1 and theCountess II FL Automated Cell Counter (with RFP
light cube) to enable accurate quantification evenin the presence of sub-cellular debris. The optimalcell concentration for the Countess is 1,000-4,000
cells/μl. Refer to manufacturer's instructions fordetails on operations.
• Vortex Ethidium Homodimer-1, centrifuge
briefly, and dilute the concentrated stock as per
manufacturer's instructions (~1:100 dilution).
• Aliquot 10 μl diluted Ethidium Homodimer-1 ina tube.
• Gently mix the sample. Immediately add 10 μl
sample to 10 μl diluted Ethidium Homodimer-1.
Gently pipette mix 10x.
• Transfer 10 μl sample to a Countess II Cell
Counting Slide chamber.

• Confirm the absence of large clumps using thebright-field mode. Make sure the cell counter iscircling RFP positive cells. Note the RFP-positive
concentration. Multiply by dilution factor 2 todetermine cell concentration.
Samples stained with Ethidium Homodimer-1 canalso be counted using Cellaca counter. Refer tomanufacturer's instructions for details.

Counting using AO/PI Staining Solution
This protocol provides instructions for counting
sample using AO/PI staining solution and theCellaca Counter to enable accurate quantificationeven in the presence of sub-cellular debris. The
optimal cell concentration for the Cellaca Counteris 100-10,000 cells/μl. Refer to manufacturer's
instructions for details on operations.
• Add 25 μl AO/PI Staining Solution into Mixing
Row of Cellaca plate• Gently mix the sample. If the sample is tooconcentrated, a 1:1 dilution in PBS can also be
prepared. For example, add 15 μl fixed cell suspension to 15 μl PBS.
• Add 25 μl sample to Mixing Row of plate containing AO/PI Staining Solution. Gently pipette mix 8x.
• Transfer stained sample to Loading Row ofCellaca plate.

• Insert the slide into the Countess II FL Cell
Counter. Image the sample using the RFP setting
for fluorescent illumination and filtering.
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8Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

Cell Yields from Fixed Dissociated Tissues
Listed below are the cell yields from 25 mg of various
mouse and human tissue types (flash-frozen or fresh)that were processed as described in this protocol. The
final cell yields are impacted by the cell density andmorphology of the tissue, and by the overall tissue
quality prior to fixation. Less than 25 mg of some of
the listed tissues may also yield sufficient cells.

Mouse

AdiposeCell Yields/25 mg tissue
Flash-frozen tissue

Fresh tissue

Not recommended
Nuclei isolation & fixation recommended

Human

Tissue Type

CG000553 | Rev B

Tissue Type

Cell Yields/25 mg tissue
Flash-frozen tissue

Jejunum

1.37 x 106

Colon*

0.04 x 106

Lung

3.4 x 106

Kidney2 x 106

Liver

1.8 x 106

Spleen9.6 x 106

Ileum

0.14 x 106

Testes2.05 x 106

Duodenum

0.32 x 106

BreastCancer

0.96 x 106

Melanoma

3.18 x 106

OvarianCancer

0.93 x 106
1.78 x 106 cells

Bladder

0.7 x 106

Not tested

Brain(Forebrain)

1 x 106

0.79 x 106

Brain(Cerebellum)

Not tested

3.1 x 10

Brain (Whole)

1.6 x 100.84 x 10

Colon

2.2 x 106

Not tested

Pancreatic
CancerEye

1.6 x 106

Not tested

Heart

0.99 x 106

2 x 106*Lower than expected yield from 25 mg colon tissue; using>25 mg colon tissue recommended for this assay.

Intestine

3.1 x 106

1.6 x 106

Kidney2.7 x 106

2.5 x 106Liver

1.3 x 106

0.57 x 106

Lung

2 x 106

3.1 x 106

Muscle0.41 x 106

Not tested

Ovary

3.2 x 106

Not tested

1.8 x 106

0.92 x 106

Pancreas

Spinal Cord

6

6

6

Digestion using Liberase TH(1 mg/ml) recommended

1.5 x 106

Not tested

High debris observed

Spleen14 x 106

6.3 x 106

Stomach2.5 x 106

Not tested

Testis4.2 x 106

Not tested

Thymus15 x 1026 x 106

Tongue1.4 x 106

Not tested

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9Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

CG000553 | Rev B

Troubleshooting
Problem
Lower than expected
cell yield after
dissociation

Solution• Increase the tissue dissociationtime.
• If using an Octo Dissociator,
after transferring the dissociatedcells from the C tube, perform
an additional PBS rinse of the
C tube and pass the rinse throughthe 30 µm strainer in step 2e tocollect additional cells.
• If using gentleMACS Octo
Dissociator, intact/large tissue
pieces at the end of run or run
fails midway. Run a "spin only"
program on the Octo Dissociatorwith steps 3-4 (from the protocol
in step 2C).
• If manually dissociating toughtissue, use the back of a 1-ml
syringe plunger to push any
undissociated tissue pieces
through the 30 µm strainer.
• If cell yield is still low, replace
Liberase TL with Liberase TH in
Dissociation solution, and increase
final Liberase concentrationfrom 0.2 mg/ml to 1 mg/ml. Ifcell yield is still low, increase thedissociation time.

References
1. Chromium Fixed RNA Profiling Reagent Kit
for Singleplexed Samples with Feature Barcode
technology for Cell Surface Protein User Guide(CG000477)
2. Chromium Fixed RNA Profiling Reagent Kit forMultiplexed Samples User Guide (CG000527)

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10Demonstrated Protocol | Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

CG000553 | Rev B

Document Revision Summary
Document Number

CG000553

Title

Tissue Fixation & Dissociation for Chromium Fixed RNA Profiling

Revision

Rev A to Rev B

Revision Date

February 2023

Specific Changes• Updated vendor recommendations for buying RPMI and Liberase TH (optional)• Updated step 1c to include tissue weights and corresponding fixation buffer volumes• Included Quenching Buffer as an option in step 1j (Fix Tissue) and step c (Fixed Cells – Post-Storage
Processing)• Updated long term fixed sample storage recommendation to −80°C• Updated to include mouse tissues tested• Updated Troubleshooting section to include additional guidance
General Changes• Updated for general minor consistency of language and terms throughout© 2023 10x Genomics, Inc. (10x Genomics). All rights reserved. Duplication and/or reproduction of all or any portion of this document without the express written consent of 10x
Genomics, is strictly forbidden. Nothing contained herein shall constitute any warranty, express or implied, as to the performance of any products described herein. Any and all warranties applicable to any products are set forth in the applicable terms and conditions of sale accompanying the purchase of such product. 10x Genomics provides no warranty andhereby disclaims any and all warranties as to the use of any third-party products or protocols described herein. The use of products described herein is subject to certain restrictions
as set forth in the applicable terms and conditions of sale accompanying the purchase of such product. A non-exhaustive list of 10x Genomics’ marks, many of which are registered in
the United States and other countries can be viewed at: www.10xgenomics.com/trademarks. 10x Genomics may refer to the products or services offered by other companies by theirbrand name or company name solely for clarity, and does not claim any rights in those third-party marks or names. 10x Genomics products may be covered by one or more of thepatents as indicated at: www.10xgenomics.com/patents. All products and services described herein are intended FOR RESEARCH USE ONLY and NOT FOR USE IN DIAGNOSTIC
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