TITLE: Single Cell Dissociation of Fresh Lung Tissue
AUTHORS: Lance Peter, Mei-I Chung, Nicholas E. Banovich

[STEPS]
?. [Lung Dissociation]
Transfer distal lung tissue to a cold petri dish and dissect into required pieces for the experiment
?. [Lung Dissociation]
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  of  Enzyme Mix.    ABC1ReagentCatalog NumberFinal Concentration2Collagenase Type ILS0041971 mg/ml3Collagenase Type 4LS0041881 mg/ml4Neutral Protease (Dispase)LS021061 mg/ml5DNase I (RNAse & Protease-free)LS00633110 ug/ml6HBSS14025-092to 7 ml
7 ml
4 °C
ABC1ReagentCatalog NumberFinal Concentration2Collagenase Type ILS0041971 mg/ml3Collagenase Type 4LS0041881 mg/ml4Neutral Protease (Dispase)LS021061 mg/ml5DNase I (RNAse & Protease-free)LS00633110 ug/ml6HBSS14025-092to 7 ml
?. [Lung Dissociation]
Place on GentleMACS Dissociator using the program for  at  .
37 °C
Program installation on GentleMACS:1. Unzip GM8.zip and place GM8 folder in a blank USB.2. With machine on, insert USB3. Select the Editor menu -> USB -> Select the program in GM8 folder -> "Save" and select desired     destination folder
?. [Lung Dissociation]
Add  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.  ABC1ReagentCatalog NumberFinal Concentration2FBS1600004410%3PBS21-040-CV1X
7 ml
4 °C
ABC1ReagentCatalog NumberFinal Concentration2FBS1600004410%3PBS21-040-CV1X
?. [Lung Dissociation]
Centrifuge 300g for  at .
4 °C
?. [Lung Dissociation]
Remove the supernatant. Resuspend the cell pellet in  1X PBS 3% BSA.  ABC1ReagentCatalog NumberFinal Concentration2BSA (7.5%)152600373%3PBS21-040-CV1X
5 ml
4 °C
ABC1ReagentCatalog NumberFinal Concentration2BSA (7.5%)152600373%3PBS21-040-CV1X
?. [Lung Dissociation]
Count cell numbers. Transfer 1 - 2 x 106 cells to a new 15 ml falcon tube.
?. [Lung Dissociation]
Centrifuge 300g for   at  . Resuspend the cell pellet in  1X PBS 3% BSA.
4 °C
1 ml
4 °C
?. [Lung Dissociation]
Add  of 50 μM calcein AM to the cell suspension.
2 µl
?. [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.    ABC1ReagentProduct Number1X (μl)2RT Reagent Mix220089503Poly-dT RT Primer20000075.94Additive A2200742.45Nuclease-free WaterNA16.76Single cell suspensionNA~15 - 16 (see next step)7RT Enzyme Mix B2000010 or 200002110 (added last)8Total100
ABC1ReagentProduct Number1X (μl)2RT Reagent Mix220089503Poly-dT RT Primer20000075.94Additive A2200742.45Nuclease-free WaterNA16.76Single cell suspensionNA~15 - 16 (see next step)7RT Enzyme Mix B2000010 or 200002110 (added last)8Total100
?. [Single Cell 5' Library ]
Immediately after incubation, FACS (Sony SH800, 70 μm sorting chip) sorting 15,000 calcein AM positive cells into collection tubes at  .
4 °C
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.
?. [Single Cell 5' Library ]
Add 10 μl of RT Enzyme Mix B to the reaction mix + cell suspension for a total of 100 μl.
?. [Lung Dissociation]
Incubate for  to  at , protected from light.
0 Room temperature
?. [Single Cell 5' Library ]
Load Chromium chip A and run Chromium controller for GEM generation.
?. [Single Cell 5' Library ]
Generate cDNA and library following the kit protocol.