Oct 28, 2019
Single Cell Dissociation of Fresh Lung Tissue
- Lance Peter1,
- Mei-I Chung1,
- Nicholas E. Banovich1
- 1Translational Genomics Research Institute, Phoenix, AZ
- Human Cell Atlas Method Development Community
External link: https://www.biorxiv.org/content/10.1101/753806v1
Protocol Citation: Lance Peter, Mei-I Chung, Nicholas E. Banovich 2019. Single Cell Dissociation of Fresh Lung Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.7xkhpkw
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: October 03, 2019
Last Modified: October 28, 2019
Protocol Integer ID: 28364
Keywords: Single Cell, Lung tissue, scRNAseq
Guidelines
All practices follow all safety guidelines regarding human tissue handling.
Materials
MATERIALS
Collagenase Type 1Worthington Biochemical CorporationCatalog #LS004197
Collagenase Type 4Worthington Biochemical CorporationCatalog #LS004188
Neutral Protease (Dispase)Worthington Biochemical CorporationCatalog #LS02106
DNase I RNAse & Protease-freeWorthington Biochemical CorporationCatalog #LS006331
HBSSGibco - Thermo Fisher ScientificCatalog #14025-092
PBS without Ca2 and Mg2CorningCatalog #21-040-CV
100um cell strainerVWR International (Avantor)Catalog #10199-658
MACS SmartStrainers 30umMiltenyi BiotecCatalog #130-098-458
GentleMACS C tubeMiltenyi BiotecCatalog #130-093-237
Bovine Albumin Fraction V (7.5% solution)Gibco - Thermo Fisher ScientificCatalog #15260037
Lung Dissociation
Lung Dissociation
Transfer distal lung tissue to a cold petri dish and dissect into required pieces for the experiment
One piece tissue (2cm3) blot dry on disposable underpad and place to another cold petri dish. Mince the tissue into smaller pieces using scalpel. Transfer minced tissue to a GentleMACS C tube containing 7 mL of 4 °C Enzyme Mix.
| Reagent | Catalog Number | Final Concentration | |
| Collagenase Type I | LS004197 | 1 mg/ml | |
| Collagenase Type 4 | LS004188 | 1 mg/ml | |
| Neutral Protease (Dispase) | LS02106 | 1 mg/ml | |
| DNase I (RNAse & Protease-free) | LS006331 | 10 ug/ml | |
| HBSS | 14025-092 | to 7 ml |
Enzyme Mix
Place on GentleMACS Dissociator using the program 
GM8.zip for 00:18:00 at 37 °C .
GM8.zip for 00:18:00 at 37 °C . Note
Program installation on GentleMACS:
1. Unzip GM8.zip and place GM8 folder in a blank USB.
2. With machine on, insert USB
3. Select the Editor menu -> USB -> Select the program in GM8 folder -> "Save" and select desired
destination folder
Add 7 mL 4 °C Inactivation Media and use a 10 ml serological pipette to titrate 5-10 times 2 ml/s. Using same pipette, pass the cell suspension through series 100 μm, 30 μm strainer into a 15 ml falcon tube.
| Reagent | Catalog Number | Final Concentration | |
| FBS | 16000044 | 10% | |
| PBS | 21-040-CV | 1X |
Inactivation Media
Centrifuge 300g for 00:05:00 at 4 °C .
Remove the supernatant. Resuspend the cell pellet in 5 mL 4 °C 1X PBS 3% BSA.
| Reagent | Catalog Number | Final Concentration | |
| BSA (7.5%) | 15260037 | 3% | |
| PBS | 21-040-CV | 1X |
1X PBS 3% BSA
Count cell numbers. Transfer 1 - 2 x 106 cells to a new 15 ml falcon tube.
Centrifuge 300g for 00:05:00 at 4 °C . Resuspend the cell pellet in 1 mL 4 °C 1X PBS 3% BSA.
Add 2 µL of 50 μM calcein AM to the cell suspension.
Incubate for 00:15:00 to 00:20:00 at Room temperature , protected from light.
Single Cell 5' Library
Single Cell 5' Library
Follow 10X Genomics Chromium Single Cell 5' Library Kit. Prepare cell collection tubes containing RT Reagent Mix, Poly-dT RT Primer, Additive A, and nuclease-free water.
CG000086_ChromiumSingleCellV_D_J_ReagentKits_UG_RevH.pdf | Reagent | Product Number | 1X (μl) | |
| RT Reagent Mix | 220089 | 50 | |
| Poly-dT RT Primer | 2000007 | 5.9 | |
| Additive A | 220074 | 2.4 | |
| Nuclease-free Water | NA | 16.7 | |
| Single cell suspension | NA | ~15 - 16 (see next step) | |
| RT Enzyme Mix B | 2000010 or 2000021 | 10 (added last) | |
| Total | 100 |
Immediately after incubation, FACS (Sony SH800, 70 μm sorting chip) sorting 15,000 calcein AM positive cells into collection tubes at 4 °C .
Note
15,000 cells is about 15 - 16 μl using Sony SH800 with a 70 μm sorting chip.
We recovered 3,000 - 4,000 cells post sequencing from loading 15,000 cells. Number of cells for loading depends on targeted cell recovery and your sample recovery efficiency. Variable factors include: sorting method, time, and nozzle diameter, 10x Chromium loading efficiency, and individual sample quality. ie. Sorting 15,000 cells (70 µm nozzle) directly to the RT buffer (minus RT Enzyme Mix B) required an additional 16.7 µl nuclease-free water to reach 100 µl total reaction volume, where using a 100 µm nozzle would require less nuclease-free water.
Add 10 μl of RT Enzyme Mix B to the reaction mix + cell suspension for a total of 100 μl.
Load Chromium chip A and run Chromium controller for GEM generation.
Generate cDNA and library following the kit protocol.