GSM3103110: cd4_3: 3'SAGE-seq; Mus musculus; RNA-Seq
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 CD8+ T cells. 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).