Jun 17, 2020
Frozen tissue dissociation for single-nucleus RNA-Seq
- 1Memorial Sloan Kettering Cancer Center
- Human Cell Atlas Method Development Community
- NCIHTAN
Protocol Citation: Linas Mazutis, Ignas Masilionis, ojasvi chaudhary 2020. Frozen tissue dissociation for single-nucleus RNA-Seq. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgb13pqvpk/v1
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: Working
We use this protocol and it's working
Created: July 18, 2019
Last Modified: June 17, 2020
Protocol Integer ID: 25981
Keywords: nuc-seq, scRNA-Seq, snRNA-Seq, frozen tissue, lung,
Abstract
The protocol described here relies on mechanical dissociation of frozen lung tissue (human or mouse), filtering and washing the nuclei suspension in SCC buffer followed by the FACS sorting. The resulting nuclei suspension can be processed on scRNA-Seq platform of choice, 10X Chromium, inDrops or other. The protocol has been validated on frozen human tissues of lung, breast pancreas and kidney as well as frozen mouse tissues of brain, lung, pancreas.
Materials
MATERIALS
Nuclei EZ lysis buffer Merck MilliporeSigma (Sigma-Aldrich)Catalog #EZ PREP NUC-101
Dounce homogenizers Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8938-1SET
Ambion RNase Inhibitior 40 U/µlAmbionCatalog #AM2682
DEPC (Diethyl pyrocarbonate)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D5758-25ML
HemocytometerThermo Fisher ScientificCatalog #A25750
CorningTM FalconTM Test Tube with Cell Strainer Snap CapThermo Fisher ScientificCatalog #352235
Plastic petri dish 35 x 10 mm styleFalconCatalog #353001
Protein LoBind tubesEppendorfCatalog #022431081
Disposable ScalpelTechnocutCatalog #10148-882
Ambion RNase Inhibitor 40U/uLAmbionCatalog #AM2682
DEPC-treated waterAmbionCatalog #AM9915G
DNAse/RNAse free distilled waterInvitrogenCatalog #10977023
10% (w/v) BSA filtered through 0.2 μm membraneMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7906-100G
DAPI (46-Diamidino-2-Phenylindole Dihydrochloride) at 1 mg/1 mLInvitrogenCatalog #D1306
Trypan Blue Solution 0.4%Catalog #15250061
SSC (20X) RNase-freeInvitrogenCatalog #AM9770
DRNA Free Reagent Spray Catalog #UX-04397-24
1M DTTMerck MilliporeSigma (Sigma-Aldrich)Catalog #43816-10ML
70% Ethanol spray
Sucrose Merck MilliporeSigma (Sigma-Aldrich)Catalog #S7903-250G
Citric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #251275-100G
Equipment
Swinging bucket cooling centrifuge
NAME
Centrifuge
TYPE
SorvallTM LegendTM X1 Centrifuge Series
BRAND
n/a
SKU
Equipment
Countess II
NAME
Life Technologies
BRAND
AMQAX1000
SKU
10% (w/v) BSA: Prepare 10% BSA solution and filter through 0.22 μm membrane (PTFE). Store at -20 ºC.
Nuclei Lysis buffer:
| Reagent | Volume, µl | Final concentration | |
| 2 M Sucrose | 125 | 250 mM | |
| 250 mM Citric Acid | 200 | 50 mM | |
| DEPC treated water | 675 | - - - |
Total volume 1.0 ml
Nuclei Wash buffer:
| Reagent | Volume, µl | Final concentration | |
| 2 M Sucrose | 125 | 250 mM | |
| 250 mM Citric Acid | 200 | 50 mM | |
| 10% (w/v) BSA | 10 | 1% (w/v) | |
| 1M DTT | 20 | 20 mM | |
| Ambion RNAse Inhibitor, 40U/ul | 5 | 0.2 U/ul | |
| DEPC treated water | 640 | - - - |
Total volume 1.0 ml
Nuclei Resuspension buffer:
| Reagent | Volume, µl | Final concentration | |
| 6X SCC | 1500 | 3X | |
| 1M DTT | 60 | 20 mM | |
| 10% (w/v) BSA | 30 | 1% (w/v) | |
| Ambion RNAse Inhibitor, 40U/ul | 15 | 0.2 | |
| DEPC treated water | 1395 | - - - |
Total volume 7.0 ml
Before start
Evaluate RNA integrity
Before you start make sure that the frozen tissue has RIN value higher than 6.0
Tissue grinding
Tissue grinding
15m
15m
Prepare the laminar hood and the grinding glassware:
- Clean the surface and glassware thoroughly with DRNAse Free Reagent Spray followed by 70% ethanol.
- Bring the ice bucket in the cell culture hood and spray it with DRNAse Free Reagent.
- Place fully assembled Dounce homogenizer on ice and to let it cool.
- Bring sterile blade in the hood.
3m
Prepare a frozen tissue:
- On a dry ice, place a frozen tissue on a Petri dish and cut it into two or three smaller pieces (e.g. the size of which corresponds to a half-rice grain).
3m
Prepare Lysis buffer:
- Transfer 600 µL Lysis Buffer in 1.5 mL tube, add 1.0 µL of DEPC and vortex vigorously until solution becomes cloudy and homogenous.
Note
DEPC is not soluble in water and it will form layer of droplets on top. Therefore, mix vigorously to disperse DEPC droplets.
- Transfer ready-to-use lysis solution in 2 mL grinding glass tube.
1m
Grind the tissue:
- Place the small piece(s) of frozen tissue in 2 mL grinding glass tube.
- Using a large clearance pestle (Tube A) for the initial sample grinding gently move pestle up and down 10-15 times.
Note
Do not remove pestle out of the liquid while grinding the tissue, avoid creating bubbles, which may form if pestle is being moved too quickly.
- Next, use a small clearance pestle (Tube B) and grind tissue further by movinig up and down for 10-15 times.
- Tissue should be completely minced and homogenized.
Note
Certain tissue types might be elastic and hard to break down completely. In such case move to next step.
- Keep the sample in a glass tube on ice.
5m
Record experimental details:
- Write down if something unusual happened, tissue did not dissociate, etc.
2m
Straining/washing of single-nuclei suspension
Straining/washing of single-nuclei suspension
25m
25m
IMPORTANT: keep nuclei suspension cool at all times.
NOTE: For centrifugation use a swinging bucket centrifugure and 1.5 ml (or 2.0 ml) Protein LoBind Eppendorf tubes placed inside the 50 mL Falcon tube.
Straining:
- Place 5 mL Falcon Round-bottom tube (typically used for FACS) with blue Snap Cap strainer on ice.
- Strain the nuclei suspension through 35 µm Cell Strainer Snap Cap and collect flow through fraction into the tube.
Note
If it is difficult to aspirate the nuclei suspension by pipette another option is to pour all suspension out of 2 mL homogenizer tube directly onto the blue cap. However, care must be taken to not introduce any ice that might be stuck on the outside walls of a homogenizer tube.
- Transfer the filtered solution to 1.5 mL Protein LoBind Eppendorf tube, keep on ice.
2m
Centrifugation:
- Spin down the dissociated tissue suspension in a swinging bucket centrifuge at 500 x g, 4°C, 00:05:00 .
- There should be a clearly visible pellet. If you don't see one, check the settings on your centrifuge.
- Remove and save supernatant from the centrifuged nuclei pellet. Do not disturb the pellet, leave ~ 10-20 uL of supernatant on top.
7m
Resuspension:
- Re-suspend the pellet in 1 mL Nuclei Wash Buffer using 1000 µL pipette.
2m
Centrifugation:
- Spin down the tube in a swinging bucket centrifuge at 500 x g, 4°C, 00:05:00 .
- Remove the supernatant without disrupting the pellet, the size of which should be smaller than in the previous step.
7m
Resuspension and straining:
- Re-suspend the nuclei pellet in 1 mL Nuclei Wash Buffer , mix well using 1000 µL pipette until suspension becomes homogenious. The volume of buffer at this step can be adjusted as needed (e.g. 0.5 ml).
- Filter the nuclei suspension through 5 mL FACS Snap Cap strainer and collect into the tube.
2m
Nuclei evaluation under BF/FL microscope:
- Mix 10 µL nuclei suspension with 0.2 µL 100X DAPI dye and 10 µL 0.4% Trypan Blue dye .
- Inspect nuclei suspension under the bright-field and fluorescence microscope. Record the images (Figure 1).
- Approximately 106- 107 nuclei per 1 ml should be expected.
Note
When evaluating sample under the bright field there should be a lot of dark debris visible (Figure 1) making it hard to count the species. However, i the blue (DAPI) channel the nuclei should be clearly visible.
Figure 1. Bright field microscopy image of nuclei suspension before FACS (blue circles indicate individual nuclei).
5m
FACS
FACS
25m
25m
Prepare the unstained nuclei sample:
- Take 50 µL nuclei suspension and transfer to another 5 mL Falcon tube.
- Add 250 µL Nuclei Resuspension Buffer .
- Label the tube as “reference” which will be used as a unstained population reference for FACS.
- Keep on ice
1m
Prepare the DAPI-stained nuclei sample:
- Take remaining, 950 µL nuclei suspension and stain with 10 µL 100X DAPI dye by mixing the suspension with 1 ml pipette.
- Keep on ice
1m
Prepare the FACS collection tube:
Add 0.2 mL Nuclei Resuspension Buffer into 5 mL Falcon tube and swirl buffer all around to wet the inner walls.
1m
Sort DAPI positive nuclei on FACS
- Run the "reference" sample (from Step 13) and record the scatter plot.
- Run DAPI-positive nuclei suspenion (from Step 14), indetify the fluorescent population and record the scatter plot.
- Using the collection tube preapred in Step 15, sort the DAPI-postiive population into the collection tube
- The example of sorting gates (P4) is provided below:
Figure 2. FACS plots and sorting-gates (P4) of DAPI stained nuclei suspension.
20m
Post-FACS
Post-FACS
10m
10m
Concentrate the nuclei
- Concentrate nuclei by transferring FACS sorted suspension in 1.5 mL Protein LoBind tube and spinning in a swinging bucket centrifuge at 600 x g, 4°C, 00:05:00 .
Note
Using FACS counts aim to reach a dilution of 2000 nuclei/ul.
Count the nuclei
- After centrifugation carefully aspirate the supernatant leaving the desirable amount of suspension (e.g. 200 µl)
- Carefully disperse the nuclei by mixing suspension with a 200 µl pipette.
- Mixi 10 µL Nuclei Suspension with 0.2 µL 100X DAPI dye and 10 µL 0.4% Trypan Blue dye and count the nuclei on Countess II instrument.
- Expected results are shown in Figure 3 and Figure 4.
Figure 3. Bright field microscopy image of lung nuclei suspension obtained after frozen tissue dissociation and FACS.
Single nucleus RNA-Seq
Single nucleus RNA-Seq
Control experiment:
- If frozen tissue dissociation is being conducted for the first time perform bulk cDNA synthesis and evaluate the cDNA yields and profile.
- Otherwise, proceed to the reverse transcription reaction using the platform and method of your choice.
Barcoding, library preparation and sequencing
Sequencing
Sequencing
Sequence the DNA library
We use NovaSeq 6000 instrument to sequence the final DNA libraries using pair-end sequencing option, R1 read - 26 cycles, R2 read - 70 cycles, and index read - 8 cycles, aiming for ∼100 million reads per ~5,000 single-nuclei.
FASQ files were processed using SEQC pipeline (Azizi et al., Cell, 2017) and mapped to reference genome with the default SEQC parameters to obtain the gene-cell count matrix.
Expected result
Frozen Human Lung - SCLC
Figure 4. snRNA-Seq of previously frozen human small cells lung carcinoma sample.
Expected result
Frozen Human Lung - cSCLC
Figure 5. snRNA-Seq of previously frozen combined small cells lung carcinoma sample.