WT_CT0
Mus musculus
Protocols: 1. RNAs from Laser capture microdissection and RNA seq analysis CCSP-Bmal1-/- and control Bmal1f/f mice were entrained 7 days in light dark cycles (7am light on and 7pm light off) before being kept in consistent darkness for 24h. Lungs were collected every 4h for 48h in dark dark conditions, 1 mouse/genotype/time point. Laser capture microdissection of Club cells was done as described before with modifications (Betsuyaku T, 2004, Mi-cron 35(4):229-34). After schedule 1 killing (pentobarbital i.p.), mouse lung was inflated with ̃0.8ml of 50% v/v of OCT/PBS solution via tracheal cannula. Dissected lung samples were frozen on dry ice and stored at -80°C. In cryostat, samples were cut into 10μm sections onto polyethylene naphthalate membrane slides (11600288, Leica Microsystems, Wetzlar, Germany). After se- rial dehydration in ethanol solutions (100%, 75%, 50%, 95%, 100% and 100%, 30s each), slides were air dried and then dissected in Leica LMD6500 (Leica Mi- crosystems, Wetzlar, Germany). Distal bronchiolar epithelium was cut, about 200nm from alveolar opening 2. RNAs from cultured cells Briefly, after overnight di- gestion of tracheas in pronase solution (0.15% w/v in mTEC basic medium, Dulbecco's modified Eagle's medium/Ham's F-12 (DMEM/F-12) supplemented with 15 mM HEPES, 4 mM L-Glutamine, 3.6 mM NaHCO3,100 U/ml penicillin, and 100 mg/mL streptomycin). Cells were seeded in collagen coated cell inserts for 24 well plates. Cells were cultured for 7 days submerged in mTEC Plus buffer (mTEC/Basic medium supplemented with 10 mg/ml insulin, 5 mg/ml transferrin, 0.1 mg/ml Cholera toxin, 25 ng/ml epidermal growth factor, 30 mg/ml bovine pituitary extract, 5% FBS (v/v)) to expend cell number. Then under air-liquid interface culture condition, they were differentiated in mTEC SF buffer (mTEC/Basic medium supplemented with 5 mg/ml insulin, 5 mg/ml transferrin, 0.025 mg/ml CT, 5 ng/ml epidermal growth factor, 30 mg/ml bovine pituitary extract, 1 mg/ml BSA) for 9 days to induce cilia genesis. Experiments were carried out in mixed males and females. In the end of experiment, cells were fixed for immunofluorescence staining or frozen for RNA extraction. RNA was purified by PicoPure RNA Isolation Kit (Thermo Fisher Scientific, Paisley, UK) with on-column Dnase digestion. RNA integrity was checked by Agilent RNA 6000 pico kit (Agilent Technolo- gies, Cheshire, UK). Poly(A) selected mRNA (100ng total RNA/sample, RIN [?] 6) was used for RNA seq. The library was constructed with TruSeq V3 kit (Illumina, California, USA) with low input method.
ENA first public
2018-12-15
ENA last update
2018-01-16
External Id
SAMEA104471046
INSDC center alias
UOM
INSDC center name
University of Manchester
INSDC first public
2018-12-15T17:02:11Z
INSDC last update
2018-01-16T16:38:49Z
INSDC status
public
Submitter Id
E-MTAB-6384:WT_CT0
age
10 to 20
broker name
ArrayExpress
cell type
respiratory epithelial cell
common name
house mouse
genotype
wild type genotype
organism part
lung
sample name
E-MTAB-6384:WT_CT0
sex
mixed
strain
C57BL/6