title: GSM8484313: postPBS_M2_RNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Alveolar type II epithelial cells (Type II pneumocytes) were isolated according to published procedures. Briefly, the lungs were perfused with 10 mL cold phosphate buffered saline (PBS) before enzymatic digestion with 2 mL of dispase infused into the lung, after which they were removed and incubated in an additional 2 mL of dispase for one hour. The lungs were then manually disintegrated and the resulting cell suspension labeled with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32 (Thermo Fisher Scientific). Samples  were pooled to include three sets of lungs per sample for a total of three samples for FACS separation. Type II pneumocytes were isolated by negative selection and thus identified as the unlabeled cell population. Type II pneumocytes were also gated as sideward scatter high (SSChigh) cell population which minimizes contamination with lymphoid cells by selecting cells with a higher granularity. The cells were separated by fluorescence activated cell sorting (FACS)  using a BD FACS Aria II P07800142 (BSL2) (BD Biosciences, San Jose, CA). Following isolation via FACS, Type II pneumocytes were pelleted by centrifugation. The samples were first centrifuged for 12 min at 200 x g and 4°C and the supernatant was removed, except for the bottom 1 mL, which was transferred to a 1.7 mL Eppendorf tube. To this tube, 500 µL of PBS was added, and the tube was centrifuged for 12 min at 200 x g at 4°C. The supernatant was removed while the bottom 100 μL was saved. One mL of PBS was added to the tube, and the sample was centrifuged for 12 min at 800 x g and 4°C. After this final centrifugation, all supernatant is removed, and the cell pellet was used to isolate DNA and RNA using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Hilden Germany) according to the man-ufacturer's instructions. Total RNA samples were converted to Illumina sequencing libraries using SMARTer Stranded Total RNA-Seq Kit – Pico Mammalian Input (Takara Bio USA, Mountain View CA). Alveolar type II epithelial cells (Type II pneumocytes) were isolated according to published procedures. Briefly, the lungs were perfused with 10 mL cold phosphate buffered saline (PBS) before enzymatic digestion with 2 mL of dispase infused into the lung, after which they were removed and incubated in an additional 2 mL of dispase for one hour. The lungs were then manually disintegrated and the resulting cell suspension labeled with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32 (Thermo Fisher Scientific). Samples  were pooled to include three sets of lungs per sample for a total of three samples for FACS separation. Type II pneumocytes were isolated by negative selection and thus identified as the unlabeled cell population. Type II pneumocytes were also gated as sideward scatter high (SSChigh) cell population which minimizes contamination with lymphoid cells by selecting cells with a higher granularity. The cells were separated by fluorescence activated cell sorting (FACS)  using a BD FACS Aria II P07800142 (BSL2) (BD Biosciences, San Jose, CA). Following isolation via FACS, Type II pneumocytes were pelleted by centrifugation. The samples were first centrifuged for 12 min at 200 x g and 4°C and the supernatant was removed, except for the bottom 1 mL, which was transferred to a 1.7 mL Eppendorf tube. To this tube, 500 µL of PBS was added, and the tube was centrifuged for 12 min at 200 x g at 4°C. The supernatant was removed while the bottom 100 μL was saved. One mL of PBS was added to the tube, and the sample was centrifuged for 12 min at 800 x g and 4°C. After this final centrifugation, all supernatant is removed, and the cell pellet was used to isolate DNA and RNA using the Qiagen AllPrep DNA/RNA Mini Kit (Qiagen, Hilden Germany) according to the man-ufacturer's instructions.