title: GSM2671494: miR20a_2_3'SAGE-seq; Mus musculus; RNA-Seq
library_construction_protocol: Transferred lung fibroblasts were isolated from bleomycin-treated lungs of C57BL/6 mice at day 3 post-transfer. Lungs were cut into small pieces with a razor blade and digested enzymatically using 0.2% collagenase (Wako, Osaka, Japan), 0.96 mg/mL Dispase II (Roche, Basel, Switzerland), and 20 kU/mL DNase I (Sigma-Aldrich, St Louis, MO) for 60 min at 37°C. Erythrocytes were removed by 70% Percoll (GE Healthcare, Buckinghamshire, UK) density separation. The single-cell suspensions were then depleted of lineage+ (CD31+, CD45+, EpCAM+, CD146+ and Ter119+) cells by negative selection with an AutoMACS cell separator (Miltenyi Biotech, Bergisch Gladbach, Germany). Lineage− Col1a2GFP+ hLNGFR+ fibroblasts were further purified by cell sorting using a FACS Aria II (BD). polyA RNAs were isolated and amplified from donor 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).
attributes.tag: GEO Accession
attributes.value: GSM2671494