title: GSM3406228: D2A1.2.P; Mus musculus; RNA-Seq
library_construction_protocol: Lungs and primary tumors were harvested from mice, immersed in PBS, and promptly chopped with scissors to small fragments. Minced lungs were then added to digestion solution (PBS buffer with 75mg/ml TM Liberase, Roche 05401151001, 75mg/ml TH Liberase, Roche 05401127001, 12.5mg/ml DNAse, Sigma-Aldrich DN25) for 1hr at 37°C, rocking. Digested lung pieces were spun down for 5' at 1300rpm, re-suspended in calcium- and magnesium-free PBS plus 1mM EDTA by vigorous pipetting until the solution was homogeneous and eventually filtered through a 70mm cell strainer to remove undigested fibrous tissue. In the case of stiffer tissues, such as primary tumors, tissue fragments were also mechanically disrupted by passing them through needles of decreasing thickness. Cells were then pelleted and red blood cells lysed with Red Blood Cells Lysis Solution (Miltenyi Biotec, 130-094-183) following manufacturer protocol. After washing, cells were re-suspended in FACS buffer (PBS, 2mM EDTA, 3%BSA) and labelled with CD45-APC antibody for 30min (eBiosciences, 30-F11, 1:400) to avoid contamination from leukocytes during sorting. Samples were then washed repeatedly, filtered through a 70mm cell strainer and kept on ice during fluorescence-activated cell sorting. Total RNA was extracted with RNeasy Plus Micro kit (Qiagen) following manufacturer protocol. RNA samples were assessed for quantity and integrity using the NanoDrop 8000 spectrophotometer V2.0 (Thermo Fisher Scientific) and Agilent 2100 Bioanalyser (Agilent Technologies), respectively. Samples displayed low levels of degradation with RNA integrity numbers (RIN) between 6.4 and 7.8. Full-length cDNA molecules were generated from 4ng of total RNA per sample using the SMARTer kit for cDNA generation (Clontech). cDNA quantity was measured using the dsDNA High-sensitivity Qubit kit with the Qubit 2.0 Fluorometer (Thermo Fisher Scientific), and were checked for quality using a D1000 ScreenTape with the Agilent 2200 Tapestation (Agilent Technologies). Libraries were prepared using the Illumina Nextera XT Sample Preparation Kit (Illumina Inc.) with an input of 150pg of cDNA per sample. Resulting libraries were checked for average fragment size using the Agilent D1000 ScreenTape, and were quantified using the Qubit dsDNA High-sensitivity reagent kit.
attributes.tag: GEO Accession
attributes.value: GSM3406228