Aug 19, 2020

Public workspaceHTAPP_Dissociation of human primary lung cancer resection to a single-cell suspension for single-cell RNA-seq V.2

DOI
dx.doi.org/10.17504/protocols.io.bhbgj2jw
  • 1Broad Institute;
  • 2Human Tumor Atlas Pilot Project;
  • 3Massachusetts General Hospital;
  • 4Dana-Farber Cancer Institute;
  • 5Human Tumor Atlas Project;
  • 6Massachusetts Institute of Technology;
  • 7Howard Hughes Medical Institute
  • NCIHTAN
Judit Jane-Valbuena
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DOI: dx.doi.org/10.17504/protocols.io.bhbgj2jw
Protocol Citation: Michal Slyper, Avinash Waghray, Julia Waldman, Isaac Wakiro, Mei-Ju Su, Sébastien Vigneau, Asaf Rotem, Bruce Johnson, Orit Rozenblatt-Rosen, Aviv Regev 2020. HTAPP_Dissociation of human primary lung cancer resection to a single-cell suspension for single-cell RNA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bhbgj2jw
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 24, 2020
Protocol Integer ID: 37960
Abstract

This protocol is used for the dissociation of human primary lung cancer resections to a single-cell suspension compatible with droplet-based single-cell RNA-Seq technology.

For the Human Tumor Atlas Pilot Project (HTAPP) it has been successfully applied to non-small cell adenocarcinoma from which it was able to capture a diversity of cell types, including malignant (epithelial) and non malignant cells such as  mesenchymal, endothelial, myeloid, and lymphoid cells. In some instances, this protocol can be combined with a protocol for CD45-cell depletion, which is used for fast depletion of immune cells for enrichment of malignant and stromal cells. 

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
ReagentACK Lysing BufferThermo Fisher ScientificCatalog #A1049201
ReagentPBS, pH 7.4Thermo Fisher ScientificCatalog #10010049
ReagentPronase from Streptomyces griseus Sigma AldrichCatalog #10165921001
ReagentDispase II (neutral protease grade II) Sigma AldrichCatalog #4942078001
ReagentCollagenase A from Clostridium histolyticum Sigma AldrichCatalog #10103578001
ReagentCollagenase Type 4 100MG Thermo Fisher ScientificCatalog #NC9836075
ReagentElastase from Porcine PancreasThermo Fisher ScientificCatalog #NC9301601
ReagentDNase I from bovine pancreas Sigma AldrichCatalog #11284932001
ReagentTrypan Blue solution 0.4%Sigma AldrichCatalog #T8154-20ML
ReagentNoyes Spring Scissors - Tungsten CarbideFine Science ToolsCatalog #15514-12
ReagentFlex-Tube® 1.5 mL PCR clean colorlessEppendorfCatalog #022364120
ReagentTips RT-LTS-A-10µL-/F/L-960/10RaininCatalog #30389226
ReagentTips RT-LTS-A-200µL-/F/L-960/10RaininCatalog #30389240
ReagentTips RT-LTS-A-1000µL-/F-768/8RaininCatalog #30389212
ReagentFalcon® 100 mm TC-treated Cell Culture Dish 20/Pack 200/Case SterileCorningCatalog #353003
ReagentFalcon® 15 mL High Clarity PP Centrifuge Tube Conical Bottom with Dome Seal Screw Cap Sterile 50/Rack 500/CaseCorningCatalog #352097
ReagentFalcon® 5 mL Round Bottom Polystyrene Test Tube with Cell Strainer Snap Cap 25/Pack 500/CaseCorningCatalog #352235
ReagentPipet-Lite LTS Pipette L-1000XLS RaininCatalog #17014382
ReagentPipet-Lite LTS Pipette L-200XLS RaininCatalog #17014391
ReagentPipet-Lite LTS Pipette L-20XLS RaininCatalog #17014392
ReagentCentrifuge 5430 R refrigerated with Rotor FA-45-30-11 incl. rotor lid keypad 120 V/50 – 60 Hz (US)EppendorfCatalog #022620601
ReagentShake n Stack™ Hybridization OvensThermo Fisher ScientificCatalog #6241
ReagentAspen Surgical™ Bard-Parker™ Protected Disposable ScalpelFisher ScientificCatalog #02-688-78
ReagentHBSS no calcium no magnesiumThermo Fisher ScientificCatalog #14170112
ReagentMACS SmartStrainers (70 µm)Miltenyi BiotecCatalog #130-098-462
ReagentMACS SmartStrainers (100 µm)Miltenyi BiotecCatalog #130-098-463
ReagentEppendorf Tubes™ 5.0 mLFisher ScientificCatalog #14-282-305
ReagentMACS SmartStrainers (30 µm)Miltenyi BiotecCatalog #130-098-458
ReagentUltraPure™ BSA (50 mg/mL)AmbionCatalog #AM2616
ReagentNanoEnTek Disposable HemocytometerWestnetCatalog #C-CHIP
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 one 50 mL conical tubes as “Supernatant 1” and one 15 mL conical tube as “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 PBS with 0.4% BSA, 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 3 mL per 25-200 mm3 resection, and may be adjusted depending on the size of the resection. Record in the table the volumes used.

ReagentStock concentration (mg/mL)Final concentration (µg/mL)Volume for 3 mL Mix (µL)Volume Prepared (µL)
HBSS--2692
Collagenase A150150030
Collagenase IV1001003
DNAse I1010030
Dispase II1010030
Elastase19.227.6
Pronase201250187.5

Sample Description and Allocation
Sample Description and Allocation
Report sample processing information. Temperature4 °C Wet Ice
Note
Sample ID:
Date:

Media Used for Transportation:
Person Processing:

Transfer sample to a Petri dish with cold PBS (or HBSS without phenol red) kept on ice to better visualize its composition. Take a picture of the resection alongside a ruler and annotate its different regions. Tumors are typically stiff and light-colored, often with dark spots in lung cancer. Necrotic regions tend to be soft and crumbly.
Temperature4 °C Wet Ice
Note
Describe Sample Appearance:
Insert Annotated Picture(s) of Sample:


Transfer the resection to a Petri dish with cold HBSS kept on ice and dissect the tumor from non-tumor tissue using a scalpel. Describe the decisions taken to dissect the sample and document them with an annotated picture.
Temperature4 °C Wet Ice
Note
Describe Tumor Dissection:

Dimensions of Dissected Tumor (mm):
Insert Annotated Picture(s) Documenting Tumor Dissection:
If required, divide the tumor using a scalpel and allocate pieces to different assays following each assay's requirements. Recommended dimensions to obtain enough cells for 10x Genomics Single-Cell RNA-seq system are 3x3x3 mm or larger. Describe the decisions taken to allocate the sample, including the dimensions of each piece, and document the allocation process with an annotated picture.
Temperature4 °C Wet Ice
Note
Describe Sample Allocation:

Dimensions of Tissue Piece Allocated (mm):
Insert Annotated Picture(s) Documenting Sample Allocation:
Tissue Dissociation
Tissue Dissociation
If the dimensions of the tumor piece are greater than 3x3x3 mm, cut into smaller fragments using safety scalpels.
Temperature4 °C Wet Ice
Transfer the tumor piece or fragments to a 5 mL Eppendorf tube containing 3 mL cold dissociation mix. Report time at which dissociation starts.
Tip: The suggested amount of 3 mL works well on 25-200 mm3 resections, but may be reduced or increased for smaller or larger resections, respectively.
Temperature4 °C Wet Ice
Note
Volume of Dissociation Mix Used (mL):

Dissociation Start Time:
Mince the tissue with spring scissors into <0.5 mm fragments for approximately 3 min.
Tip: If spring scissors are not available, scalpels can be used instead. In this case, the sample should be minced in a Petri dish within 1 mL dissociation mix before transferring the resulting tissue fragment suspension to a 5 mL Eppendorf tube containing an additional 2 mL of dissociation mix.
Temperature20 °C Proceed quickly at room temperature
Incubate for 10 minutes at 37˚C, with rotation at approximately 14 rpm.
Tip: Wrap the lid of the Eppendorf tube with Parafilm to prevent any leakage, and use lab tape to secure tubes on the rotator.
Duration00:10:00 Enzymatic Dissociation, Part 1
Temperature37 °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 at this step 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.
Temperature20 °C Proceed quickly at room temperature
Incubate for another 10 minutes at 37˚C, with rotation at approximately 14 rpm.
Tip: Wrap the lid of the Eppendorf tube with Parafilm to prevent any leakage, and use lab tape to secure tubes on the rotator.
Duration00:10:00 Enzymatic Dissociation, Part 2
Temperature37 °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.
Temperature20 °C Proceed quickly at room temperature
Filter through a 70 µm cell strainer set on a 15 mL conical tube kept on ice to get rid of fragments. Wash strainer with 10 mL cold HBSS.
Tip: If the mixture appears to have a lot of undigested tissue pieces or mucus, which may clog the 70 µm strainer, use a 100 µm strainer instead.
Temperature4 °C Wet Ice
Centrifuge at 400 g for 5 minutes in 4˚C pre-cooled centrifuge. This long spin helps to get rid of fat more efficiently than subsequent short spins.
Duration00:05:00 Centrifugation
Temperature4 °C Centrifuge
Red Blood Cell Removal
Red Blood Cell Removal
Report pellet color (e.g., red, pink, white) in the first row of the table at Step 20.
Temperature4 °C Wet Ice
Carefully transfer supernatant to the 50 mL “Supernatant 1” tube kept on ice, making sure to remove any fat and to not disturb the pellet.
Tip: If any fat is visible, aspirate and discard it before transferring the remaining of the supernatant using a different pipette tip.
Temperature4 °C Wet Ice

Resuspend pellet in 300-500 µL cold ACK Lysing Buffer to lyse red blood cells. If cells are in a 15 mL tube, transfer them to a 1.7 mL Eppendorf tube kept on ice.
Tip: The volume of ACK should be adjusted to the size and color of the pellet and may be increased up to 1 mL if the pellet is large or extremely bloody.
Temperature4 °C Wet Ice
Incubate for 1 minute on ice.
Duration00:01:00 ACK Red Blood Cell Lysis
Temperature4 °C Wet Ice
Mix in a volume of PBS equal to twice the volume ACK Lysing Buffer. 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.
Temperature4 °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.
Duration00:00:08 Centrifugation
Temperature4 °C Centrifuge
If the pellet is pink or red, revealing a significant portion of red blood cells, repeat steps 15-19. For each round of ACK Lysing Buffer treatment, report the ACK Lysing Buffer volume, treatment duration, and pellet color in the table below.
Tip: Avoid exceeding three rounds of 1 min ACK Lysing Buffer treatment, as this may result in important loss of cell viability.
Go togo to step #14 if pink or red pellet

RepeatACK Lysing Buffer Volume (µL)ACK Lysing Buffer Treatment Duration (min)Pellet Color
0NANA

Carefully transfer supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the pellet.
Temperature4 °C Wet Ice
Resuspend in 50 µL cold PBS with 0.4% BSA.
Temperature4 °C Wet Ice
Quality Control
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 ControlQuality Control after Optional Debris and Cell Clumps Removal (Step 25)
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 25):
[Optional] Debris and Cell Clumps Removal
[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 in 500 µL cold PBS with 0.4% BSA.
Temperature4 °C Wet Ice
Filter through 35 µm FACS tube cell strainer.
Tip: Alternately, 30 µm MACS SmartStrainer can be used to minimize cell loss.
Temperature4 °C Wet Ice
Wash filter with an additional 500 µL of cold PBS with 0.4% BSA to recover as many cells as possible.
Temperature4 °C Wet Ice
Transfer to Eppendorf tube.
Temperature4 °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.
Duration00:00:08 Centrifugation
Temperature4 °C Centrifuge
Carefully transfer supernatant to the 15 mL “Supernatant 2” tube kept on ice without disturbing the pellet.
Temperature4 °C Wet Ice
Resuspend in 50 µL cold PBS with 0.4% BSA.
Temperature4 °C Wet Ice
Repeat steps 23-24.
Go togo to step #23 Quality Control
Loading on 10x Genomics Single-Cell RNA-seq system
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%.
Temperature4 °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: