Aug 02, 2020
Version 2
HTAPP_Dissociation of human metastatic breast cancer core needle biopsy to a single-cell suspension for single-cell RNA-seq V.2
- Michal Slyper1,2,
- Julia Waldman1,2,
- Jingyi Wu3,2,
- Abhay Kanodia3,2,
- Sébastien igneau3,
- Asaf Rotem3,2,
- Bruce Johnson3,2,
- Orit Rozenblatt-Rosen1,2,
- Aviv Regev1,4,5,6
- 1Broad Institute;
- 2Human Tumor Atlas Pilot Project;
- 3Dana-Farber Cancer Institute;
- 4Human Tumor Atlas Project;
- 5Massachusetts Institute of Technology;
- 6Howard Hughes Medical Institute
- NCIHTAN
Protocol Citation: Michal Slyper, Julia Waldman, Jingyi Wu, Abhay Kanodia, Sébastien igneau, Asaf Rotem, Bruce Johnson, Orit Rozenblatt-Rosen, Aviv Regev 2020. HTAPP_Dissociation of human metastatic breast cancer core needle biopsy to a single-cell suspension for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bhbjj2kn
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: June 09, 2020
Last Modified: August 02, 2020
Protocol Integer ID: 37963
Keywords: single-cell RNA-seq, biopsy, dissociation, breast cancer, human metastatic breast cancer core needle biopsy, processing other tumor, human tumor atlas pilot project, metastatic breast cancer, cell rna, cell suspension compatible with droplet, htapp-dissociation, rna, cell suspension, lymph node, seq technology
Abstract
This protocol is used for the dissociation of human metastatic breast cancer core needle biopsies to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology (Slyper et al).
For the Human Tumor Atlas Pilot Project (HTAPP) it has been successfully applied to biopsies collected from breast, liver and lymph node.
Description of this protocol and guidance for testing and selecting methods for processing other tumor and sample types can be found in Slyper et al.
Guidelines
- Keep sample on ice and use cold reagents unless noted otherwise.
- Report information as indicated in the protocol.
Materials
MATERIALS
RPMI 1640 MediumThermo Fisher ScientificCatalog #11875093
TrypLEThermo Fisher ScientificCatalog # 12604013
ACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
Liberase™ TM Research Grade Merck MilliporeSigma (Sigma-Aldrich)Catalog #5401119001
PBS, pH 7.4Thermo Fisher ScientificCatalog #10010049
DNase I from bovine pancreasMerck MilliporeSigma (Sigma-Aldrich)Catalog #11284932001
Noyes Spring Scissors - Tungsten CarbideFine Science ToolsCatalog #15514-12
Flex-Tube® 1.5 mL PCR clean colorlessEppendorfCatalog #022364120
Tips RT-LTS-A-10µL-/F/L-960/10RaininCatalog #30389226
Tips RT-LTS-A-200µL-/F/L-960/10RaininCatalog #30389240
Tips RT-LTS-A-1000µL-/F-768/8RaininCatalog #30389212
Falcon® 100 mm TC-treated Cell Culture Dish 20/Pack 200/Case SterileCorningCatalog #353003
Falcon® 15 mL High Clarity PP Centrifuge Tube Conical Bottom with Dome Seal Screw Cap Sterile 50/Rack 500/CaseCorningCatalog #352097
Falcon® 5 mL Round Bottom Polystyrene Test Tube with Cell Strainer Snap Cap 25/Pack 500/CaseCorningCatalog #352235
Pipet-Lite LTS Pipette L-1000XLS RaininCatalog #17014382
Pipet-Lite LTS Pipette L-200XLS RaininCatalog #17014391
Pipet-Lite LTS Pipette L-20XLS RaininCatalog #17014392
Centrifuge 5430 R refrigerated with Rotor FA-45-30-11 incl. rotor lid keypad 120 V/50 – 60 Hz (US)EppendorfCatalog #022620601
Shake n Stack™ Hybridization OvensThermo Fisher ScientificCatalog #6241
Aspen Surgical™ Bard-Parker™ Protected Disposable ScalpelFisher ScientificCatalog #02-688-78
Eppendorf Tubes™ 5.0 mLFisher ScientificCatalog #14-282-305
Nunc CryoTube VialsThermo Fisher ScientificCatalog #375418
MACS SmartStrainers (30 µm)Miltenyi BiotecCatalog #130-098-458
UltraPure™ BSA (50 mg/mL)Thermo Fisher ScientificCatalog #AM2616
NanoEnTek Inc. Disposable HemocytometerNanoEnTek Inc.Catalog #C-CHIP
Troubleshooting
Safety warnings
Follow general lab safety and institutional guidelines for working with human samples and sharps.
Before start
- Set centrifuge to 4˚C.
- Set hybridization oven with rotator to 37˚C.
- Label two 15 mL conical tubes as “Supernatant 1” and “Supernatant 2” and keep on ice. These tubes will be used to collect supernatant before and after red blood cell removal, respectively, and prevent accidental loss of cells. If needed, cells can be recovered from the supernatants by centrifugation using settings from the protocol.
- Store ACK Lysing Buffer at 4˚C or cool down on ice.
- Prepare RPMI 1640 with 10% FBS and keep on ice. This solution can be prepared in advance and stored at 4˚C for several weeks.
- Prepare PBS with 0.4% BSA and keep on ice. This solution can be prepared in advance and stored at 4˚C for several weeks.
- Prepare dissociation mix immediately before use as described in the table below and keep on ice. The suggested amount is 1 mL per core for 18 gauge (~0.8 mm diameter), 10-20 mm long cores, and may be adjusted depending on the size and number of cores. Record in the table the volumes used.
| Reagent | Stock concentration (mg/mL) | Final concentration (µg/mL) | Volume for 1 mL Mix (µL) | Volume Prepared (µL) | |
| RPMI 1640 | - | - | 950 | ||
| DNAse I | 10 | 100 | 10 | ||
| Liberase TM | 2.5 | 100 | 40 |
Sample Description
Report sample processing information.
4 °C Wet Ice
Note
Sample ID:
Date:
Anatomical Site of the Biopsy:
Number of Biopsy Cores:
Core(s) Priority Number:
Media Used for Transportation:
Person Processing:
Transfer the sample to a Petri dish with cold PBS (or RPMI without phenol red) kept on ice in order to better visualize its composition. Take a picture of each core alongside a ruler, and describe its appearance (e.g., is it fragmented or necrotic?).
4 °C Wet Ice
Note
Describe Sample Appearance:
Core(s) Dimensions (mm):
Insert Picture(s) of Sample:
Tissue Dissociation
Transfer the sample to a 2 mL screw cap tube (e.g., cryotube) or Eppendorf tube containing 1 mL cold dissociation mix per core. Report time at which dissociation starts.
Tip: The suggested amount of 1 mL per core works well on 18 gauge (~0.8 mm diameter), 10-20 mm long cores, but may be reduced or increased for smaller or larger cores, respectively. If the volume of dissociation mix is larger than 1.5 mL, dissociation may be performed in a 5 mL (instead of 2 mL) Eppendorf tube.
4 °C Wet Ice
Note
Volume of Dissociation Mix Used (mL):
Dissociation Start Time:
Mince the sample with spring scissors to <0.5 mm fragments for approximately 3 min.
Tip: If spring scissors are not available, scalpels can be used instead. In this case, the biopsy should be minced in a Petri dish within <1 mL dissociation mix before transferring the resulting tissue fragment suspension to a 2 mL screw cap vial.
20 °C Proceed quickly at room temperature
Incubate for 10 minutes at 37˚C, with rotation at approximately 14 rpm.
Tip: If using an Eppendorf tube, wrap the tube's lid with Parafilm to prevent any leakage. Use lab tape to secure tubes on the rotator.
00:10:00 Enzymatic Dissociation, Part 1
37 °C Hybridization Oven, 14 rpm
Continue dissociation by pipetting up and down 20-30 times using a pipette with 1 mL tip.
Tip: Do not put the tube on ice as repeated, drastic changes in temperature may be detrimental to cell viability. Pipette down pushing against the wall of the tube for optimal dissociation. If fragments are too large for pipetting, continue mincing with spring scissors before pipetting.
20 °C Proceed quickly at room temperature
Incubate for another 10 minutes at 37˚C, with rotation at approximately 14 rpm.
Tip: If using an Eppendorf tube, wrap the tube's lid with Parafilm to prevent any leakage. Use lab tape to secure tubes on the rotator.
00:10:00 Enzymatic Dissociation, Part 2
37 °C Hybridization Oven, 14 rpm
Continue dissociation by pipetting up and down 20-30 times using a pipette with 1 mL tip. No or only very small fragments should be visible by eye and the solution should appear cloudy.
Tip: Pipette down pushing against the wall of the tube for optimal dissociation. If fragments are too large for pipetting, continue mincing with spring scissors before pipetting.
20 °C Proceed quickly at room temperature
Centrifuge at 300 g for 4 minutes in 4˚C pre-cooled centrifuge. This long spin helps to get rid of fat and debris more efficiently than subsequent short spins.
00:04:00 Centrifugation
4 °C Centrifuge
Red Blood Cell Removal
Report the cell pellet color (e.g., red, pink, white) in the first row of the table at Step 16.
4 °C Wet Ice
Carefully transfer the supernatant to the 15 mL “Supernatant 1” tube kept on ice, making sure to remove any fat and to not disturb the cell pellet.
Tip: If any fat is visible, aspirate and discard it before transferring the remaining of the supernatant using a different pipette tip.
4 °C Wet Ice
Resuspend the cell pellet in 200-500 µL cold ACK Lysing Buffer to lyse red blood cells.
Tip: The volume of ACK should be adjusted to the size and color of the cell pellet and may be increased up to 1 mL if the cell pellet is large or extremely bloody.
4 °C Wet Ice
Incubate for 1 minute on ice.
00:01:00 ACK Red Blood Cell Lysis
4 °C Wet Ice
Mix in a volume of PBS equal to twice the volume of ACK Lysing Buffer and proceed quickly to the next step.
Tip: If more than 500 µL ACK Lysing Buffer was used, mix in the largest volume of PBS that can safely fit in a 1.7 mL Eppendorf tube (500 µL - 1 mL) and proceed quickly to the next step.
4 °C Wet Ice
Centrifuge for 8 sec at 4˚C using short spin setting, with centrifugal force ramping up to (but not exceeding) 11,000 g. Proceed quickly to the next step.
Tip: Do not spin for a longer duration or at a higher centrifugal force, as this would result in cell death.
00:00:08 Centrifugation
4 °C Centrifuge
If the cell pellet is pink or red, revealing a significant portion of red blood cells, repeat steps 11-15. For each round of ACK Lysing Buffer treatment, report the ACK Lysing Buffer volume, treatment duration, and cell pellet color in the table below.
Tip: Avoid exceeding three rounds of 1 min ACK Lysing Buffer treatment, as this may result in loss of cell viability.
if pink or red pellet
| Repeat | ACK Lysing Buffer Volume (µL) | ACK Lysing Buffer Treatment Duration (min) | Cell Pellet Color | |
| 0 | NA | NA | ||
Cell Clump Dissociation
Carefully transfer the supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the cell pellet.
4 °C Wet Ice
Resuspend the cell pellet in 100 µL TrypLE per core.
4 °C Wet Ice
Hold tube in hand for 1 min while constantly pipetting up and down using a pipette with 200 µL tip.
Tip: Pipette down pushing against the wall or bottom of the tube for optimal dissociation.
00:01:00 TrypLE Treatment
Inactivate TrypLE by mixing with 200 µL of cold RPMI 1640 with 10% FBS per 100 µL TrypLE.
4 °C Wet Ice
Centrifuge for 8 sec at 4˚C using short spin setting, with centrifugal force ramping up to (but not exceeding) 11,000 g. Proceed quickly to the next step.
Tip: Do not spin for a longer duration or at a higher centrifugal force, as this would result in cell death.
00:00:08 Centrifugation
4 °C Centrifuge
Carefully transfer the supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the cell pellet.
4 °C Wet Ice
Resuspend the cell pellet in 50 µL cold PBS with 0.4% BSA.
4 °C Wet Ice
Quality Control
Mix 5 µL of single-cell suspension with 5 µL Trypan blue and load on hemocytometer.
Count and report the number of viable single cells, dead single cells, cell doublets or clumps, and whether debris are present, then calculate additional Quality Control metrics below. Take picture if possible.
| Initial Quality Control | Quality Control after Optional Debris and Cell Clumps Removal (Step 26) | ||
| Number of Viable Single Cells Counted | |||
| Number of Dead Single Cells Counted | |||
| Number of Cell Clumps or Doublets Counted | |||
| Concentration of Viable Single Cells (cells/µL) | |||
| Concentration of Dead Single Cells (cells/µL) | |||
| Concentration of Cell Clumps or Doublets (doublets/µL) | |||
| Volume of Single Cell Suspension (µL) | |||
| Total Number of Viable Single Cells | |||
| Proportion of Single Cells that are Viable (%) | |||
| Proportion of Cell Clumps or Doublets (%) | |||
| Description of debris (if any) |
Note
Insert Picture for Initial Quality Control:
Insert Picture for Final Quality Control (if additional cleanup was performed as described in Step 26):
[Optional] Debris and Cell Clumps Removal
If the quantity of debris or cell clumps is too high to load on 10x Genomics Single-Cell RNA-seq system and the number of cells is at least double from what is required to load, strain the sample as described below.
Tip: Samples should typically have less than 5% cell clumps. If enough cells (e.g., at least four times the number of cells to be loaded), strain only half of the sample and keep the other half as a backup.
Resuspend the cell pellet in 500 µL cold PBS with 0.4% BSA.
4 °C Wet Ice
Filter through a 30 µm MACS SmartStrainer into a 15 mL tube.
Tip: To maximize cell recovery, pipette residual liquid from underneath the strainer and transfer to the tube. Flow cytometry tubes with 35 µm cell strainer snap cap can also be used but typically result in lower cell recovery.
4 °C Wet Ice
Wash filter with an additional 500 µL of cold PBS with 0.4% BSA to recover as many cells as possible.
4 °C Wet Ice
Transfer to Eppendorf tube.
4 °C Wet Ice
Centrifuge for 8 sec at 4˚C using short spin setting, with centrifugal force ramping up to (but not exceeding) 11,000 g. Proceed quickly to the next step.
Tip: Do not spin for a longer duration or at a higher centrifugal force, as this would result in cell death.
00:00:08 Centrifugation
4 °C Centrifuge
Carefully transfer the supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the cell pellet.
4 °C Centrifuge
Resuspend the cell pellet in 50 µL cold PBS with 0.4% BSA.
4 °C Centrifuge
Repeat steps 24-25.
Quality Control
Loading on 10x Genomics Single-Cell RNA-seq System
If necessary, adjust the concentration before proceeding to loading on 10x Genomics Single-Cell RNA-seq system, following 10x Genomics recommendations.
Tip: 8,000-10,000 live cells are typically loaded per channel. Optimal cell recovery is achieved for concentrations between 800 and 1,200 cells/µL but deviations from that range are acceptable (see 10x Technical Note on this topic). Furthermore, it is recommended that viability be higher than 60% and the proportion of cell clumps lower than 5%.
4 °C Wet Ice
Report the information listed below about loading on 10x Genomics Single-Cell RNA-seq system, including the number and concentration of cells per channel.
Note
Time of Loading:
Person Loading:
Single-Cell RNA-seq Kit Used:
Concentration of Viable Cells Loaded (cells/µL):
Number of Cells Loaded per Channel:
Number of Channels Loaded: