GSM2671487: SiO2d7_smallRNA-seq; Mus musculus; miRNA-Seq
Primary lung fibroblasts were isolated from lungs of untreated, bleomycin-treated and silica-treated Col1a2-GFP mice. 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 and silica particles were removed by 70% Percoll (GE Healthcare, Buckinghamshire, UK) density separation. The single-cell suspensions were then depleted of lineage+ (CD31+, CD45+, EpCAM+ and Ter119+) cells by negative selection with an AutoMACS cell separator (Miltenyi Biotech, Bergisch Gladbach, Germany). Lineage− GFP+ fibroblasts were further purified by cell sorting using a FACS Aria II (BD). Small RNAs were isolated from lineage- GFP+ fibroblasts using a mirVana miRNA isolation kit (Thermo Fisher Scientific, Waltham, MA). Small RNA libraries were constructed using Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) according to the manufacturer’s instructions. The samples were sequenced twice on an Ion PGM™ System and an Ion PGM™ Sequencing 200 Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions.
GEO Accession
GSM2671487