GSM1973149: MacB3-2wk, replicate 3; Mus musculus; RNA-Seq
Tumor-bearing mice were sacrificed at 2 and at 3 weeks after cancer cell injection, together with control uninjected mice. The lung circulation was perfused with PBS/heparin (20 U/ml, Sigma). Left lung lobes containing tumors were harvested. For control mice, all the lung lobes were used. Tissues from 3-5 mice were pooled, mechanically dissociated and incubated for 30 min at 37°C with 3.2 mg/ml Collagenase Type 2 (Worthington, 43C14117B), 0.75 mg/ml Elastase (Worthington, 33S14652), 0.2 mg/ml Soybean Trypsin Inhibitor (Worthington, S9B11099N), and DNase I 40µg/ml (Sigma). During incubation, samples were placed in a shaking water bath and dispersed by pipetting every 10 minutes. Resulting single-cell suspensions were filtered through 70 um cell strainers (BD Biosciences) and washed with staining buffer (PBS containing 1% FBS, 2mM EDTA, 10mM HEPES). Samples were subject to red blood cell lysis, washed with staining buffer and filtered through 40 um cell strainers (BD Biosciences). Prior to staining, FcgammaR was blocked with anti-CD16/CD32 antibody (BD Biosciences) for 10 minutes. Cells were stained for 30-45 min at 4°C with the following antibodies: CD11b-FITC (Clone M1/70, BD Biosciences), SiglecF-PE or AlexaFluor647 (Clone E50-2440, BD Biosciences), Ly6G-PE-Cy7 (Clone 1A8, BD Biosciences), CD64-PE or AlexaFluor647 (Clone X54-5/7.1, BD Biosciences), CD11c-APC-Cy7 (Clone HL3, BD Biosciences), CD11c-PerCP-Cy5.5 (Clone N418, BioLegend). Flow cytometry-based cell sorting was conducted using XDP-100 or Astrios (Beckman Coulter) cell sorters. The sorting strategy involved excluding debris and cell doublets by light scatter, and dead cells by DAPI (1 ug/ml). Total RNA was isolated from flow cytometry-sorted cells using the QIAshredder and RNeasy Plus Micro kit (Qiagen). RNA quality and quantity was analyzed using NanoDrop and Bioanalyzer. Libraries were constructed using NuGen Ovation Human FFPE RNA-Seq Multiplex System kit customized with mouse specific oligos for rRNA removal.