GSM3559597: normal lung rep3; Mus musculus; RNA-Seq
Lung metastases were induced by tail vein injection of 1 x 106 E0771-LG to syngenic C57BL6/J female mice (6-8 wk old). Lungs were perfused with PBS through the right ventricle, dissected and minced. Lung metastases (<1 mm in diameter) were dissected with aid of a dissection microscope and pooled from 2-4 mice. The tissues were then digested with an enzyme mix of Liberase DL (Sigma-Aldrich 5466202001, 0.52 U/mL) and TL (Sigma-Aldrich 5401020001, 0.26 U/mL) and DNase I (Sigma-Aldrich DN25, 150 µg/mL) diluted in basal DMEM medium with rotation for 30 min at 37°C and filtered (70-µm member). Transcription inhibitors alpha-amanitin (Sigma-Aldrich A2263, 5 µg/mL) and actinomycin D (Sigma-Aldrich A1410, 1 µg/mL) were also added in the digestion buffer. Red blood cells were removed by incubation with RBC lysis buffer (Biolegend, 420301) for 5 min on ice (once for tissues and twice for blood). Cells were stained with DAPI to distinguish live/dead cells and were blocked with anti-mouse CD16/CD32 antibody (BD Biosciences 553141) for 10 min on ice, followed by antibody staining. Target cells were sorted directly into the Extraction Buffer in the Picopure RNA isolation kit (Arcturus KIT0202) for isolation. RNA sequencing of mouse total RNA was performed at Beijing Genomic Institute, using the Ovation® RNA-Seq System V2 kit for library construction, pair-end 100 bp and Hiseq 4000, which generated 60-80M reads per sample.
GEO Accession
GSM3559597