GSM3098237: FB_P28_1 3'SAGE-seq; Mus musculus; RNA-Seq
Epithelial cells and fibroblasts from various developmental stages (E18.5, P0.5, P2, P7, P28, and P56) were isolated from C57BL/6 mice. Lungs were cut into small pieces and digested enzymatically using collagenase (Wako, Osaka, Japan), Dispase II (Roche, Basel, Switzerland), and DNase I (Sigma-Aldrich, St Louis, MO) for 60 min at 37°C. Single-cell suspended lung cells were first stained with anti-mouse CD31, CD45, Ter119, CD146, and Epcam antibodies. Cells were next stained with Streptavidin-APC, followed by anti-APC microbeads (Miltenyi). Labeled cells were magnetically depleted. Finally, lineage (CD31, CD45, Ter119, and CD146)– propidium iodide– GFP– Epcam+ live epithelial cells and lineage (CD31, CD45, Ter119, and CD146)– propidium iodide– Epcam– GFP+ fibroblasts were sorted with a MoFlo Astrios flow cytometer (Beckman Coulter). polyA RNAs were isolated and amplified from donor epithelial cells and fibroblasts according to the protocol described in (Huang H, Nucleic Actd Res, 2014) with some modifications. 3'SAGE-seq libraries were constructed according to the protocol described in (Matsumura H, Methods Mol Biol, 2012) with some modifications. Sequencing was performed by using Ion Hi-Q Chef Kit, Ion PI v3 Chip Kit and Ion Proton Sequencer (Thermo Fisher Scientific) according to the manufacture's instructions except the input library concentration (100 pM) and cycle number (200).
GEO Accession
GSM3098237