Expression profiling by high throughput sequencing
Summary
To profile airway and parenchyma transcriptomes in our mouse model of asthma chronic obstructive pulmonary disease overlap, we employed RNA-Seq (TruSeq Stranded mRNA sample preparation, NovaSeq 6000, 20 million 100 bp single reads) as a discovery tool to identify mRNAs of interest in the development of experimental asthma chronic obstructive pulmonary disease overlap. Experimental asthma: Some mice were sensitized and challenged intranasally with house dust mite (HDM) extract 5 days per week for 3 weeks. In other mice, this was HDM treatment 3 times/week from weeks 4-11. Controls received phosphate buffered saline (PBS). Experimental chronic obstructive pulmonary disease (COPD): Mice were exposed to cigarette smoke (CS) twice per day, 5 days per week for 8 weeks. Control mice were exposed to normal air (AIR). Experimental asthma-COPD overlap (ACO): Mice were sensitized and challenged intranasally with HDM or PBS. From weeks 4-11, mice were also exposed to CS or AIR. Treatment with dexamethasone (DEX): Separate groups of mice were treated with DEX during week 11. Lungs were excised, airway and parenchyma tissue were isolated through blunt dissection, and total RNA isolated.
Overall design
Experimental asthma: Some mice were sensitized and challenged intranasally with house dust mite (HDM) extract 5 days per week for 3 weeks. In other mice, this was HDM treatment 3 times/week from weeks 4-11. Controls received phosphate buffered saline (PBS). Experimental chronic obstructive pulmonary disease (COPD): Mice were exposed to cigarette smoke (CS) twice per day, 5 days per week for 8 weeks. Control mice were exposed to normal air (AIR). Experimental asthma-COPD overlap (ACO): Mice were sensitized and challenged intranasally with HDM or PBS. From weeks 4-11, mice were also exposed to CS or AIR. Treatment with dexamethasone (DEX): Separate groups of mice were treated with DEX during week 11.
In total with all of the combinations above there are 10 treatment groups, with each group having 2 samples (airway and parenchyma). Below is the breakdown of all groups sequenced: 1. 3 week PBS airway 2. 3 week PBS parenchyma 3. 3 week HDM airway 4. 3 week HDM parenchyma 5. 11 week PBS AIR airway 6. 11 week PBS AIR parenchyma 7. 11 week PBS AIR DEX airway 8. 11 week PBS AIR DEX parenchyma 9. 11 week HDM AIR airway 10. 11 week HDM AIR parenchyma 11. 11 week HDM AIR DEX airway 12. 11 week HDM AIR DEX parenchyma 13. 11 week PBS CS airway 14. 11 week PBS CS parenchyma 15. 11 week PBS CS DEX airway 16. 11 week PBS CS DEX parenchyma 17. 11 week HDM CS airway 18. 11 week HDM CS parenchyma 19. 11 week HDM CS DEX airway 20. 11 week HDM CS DEX parenchyma
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