GSM8819544: Lung_WT4; Mus musculus; RNA-Seq
Lungs and liver harvesting were performed after perfusion with ice-cold PBS through the heart, organs were placed into 5 ml tubes containing 2 ml digestion solution with 2mg/ml Collagenase IV (ThermoFisher) and 0.1mg/ml DNAse I (Sigma) and cut into small pieces with scissors. The solution was incubated in a shaker at 37°C for 1 hour. Tissues were further homogenized using a 20G x 1” needle and a 5 ml syringe, and homogenates were filtered through a 100μm cell strainer. Red blood cells (RBC) from lungs and liver were lysed with ACK for 2 min, washed with PBS 1% FCS, 2mM EDTA (FACS buffer), and kept on ice until flow cytometry staining.  Peritoneal macrophages were obtained by peritoneal lavage and kept on ice until flow cytometry staining FACS was performed on a BD FACS Aria III (BD Biosciences) to achieve > 95% purity. Dead cells were excluded by live/dead viability dye (Thermo) staining. RNAseq was obtained by sorting 5000–100000 cells from each population directly into RLT buffer (Qiagen) containing 1% 2-ME and submitted to the Emory Integrated Genomics Core at Emory University (Atlanta, GA).Total RNA was isolated using the Quick-RNA Microprep Kit (Zymo Research). The cDNA was generated using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara Bio), and sequencing libraries were generated from the cDNA using the Nextera XT kit (Illumina). Libraries were pooled at equimolar ratios and sequenced on a Novaseq 6000 at approximately 100M reads per sample using 100PE reads.