alias: E-MTAB-6384:KO_CT16_p
title: Illumina HiSeq 2500 paired end sequencing; RNASeq of Circadian clock genes form wild type and Bmal1 knockout mice of airway epithelial cells
design: RNASeq of Circadian clock genes form wild type and Bmal1 knockout mice of airway epithelial cells
library_construction_protocol: 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.
identifiers.namespace: UOM
identifiers.id: E-MTAB-6384:KO_CT16_p
identifiers.namespace: University of Manchester
identifiers.id: E-MTAB-6384:KO_CT16_p
attributes.tag: Experimental Factor: genotype
attributes.value: Bmal1 knockout
attributes.tag: Experimental Factor: cell type
attributes.value: respiratory epithelial cell
attributes.tag: Experimental Factor: time
attributes.value: 16