GSM6600616: CD11c+ lung cells pooled from 2 untreated female mice; Mus musculus; RNA-Seq
Lungs were excised from four naive mice (two males and two females) into DMEM media supplemented with HEPES (Gibco), Penicillin, Streptomycin and L-Glutamine (Life Technologies). For lung digestion, 25 ug of DNase I (Sigma) and 0.28 Wunitz Units of Liberase TM (Roche) were added to each mL of digest. Lungs were snipped into small pieces with scissors in a 1.7 mL eppendorf tube and incubated on a 37°C shaker for 30-45 min at 700 rpm on a ThermoMixer F1.5 (Eppendorf). The digestion reaction was quenched by adding FBS and EDTA to a final concentration of 5% FBS and 10 mM EDTA. Digested lungs were then gently dissociated and passed through a 70 um strainer to yield single cell suspensions that were then centrifuged and resuspended in DMEM supplemented with 5% FBS, HEPES, Penicillin, Streptomycin and L-Glutamine. Single cell suspensions of two lung samples from mice of the same sex were pooled and washed with a 2X volume of ACK Lysing Buffer (Quality Biological) as above to lyse red blood cells and then were resuspended in PBS with 0.5% BSA (Roche) and 2 mM EDTA (Gibco) (MACS buffer). Cells were depleted of B cells and partially depleted of AMs and cells that had lost membrane integrity by first incubating with anti-IgD Biotin (Southern Biotech) and anti-Siglec-F Biotin, as well as 0.25 mM of NHS-Biotin (Life Technologies, freshly reconstituted in PBS), for 30 min on ice. Cells were washed in MACS buffer and incubated with 50 ml of MyOne Streptavidin T1 Dynabeads (Thermo Fisher) for 30 min, during which tubes were inverted and rotated at 4°C. Tubes were placed in a Dynamag-2 magnet (Thermo Fisher) for 2 min for bead isolation and the negative fraction containing unlabeled cells was collected and diluted with MACS buffer to 5 ml. To further enrich for macrophages and DCs and to remove stromal cells, 3ml of 14% OptiprepTM (Axis-Shield) was layered below the cell suspension, and cells were centrifuged at 600 g for 20 min with no brake at 8°C. Cells at the interface were collected and washed in MACS buffer. Finally, CD11c+ cells were enriched by incubating with CD11c Mojosort Nanobeads (Biolegend) for 15 min at 4°C. Bead-labeled cells were purified with the Mojosort magnet (Biolegend) according to the manufacturer's instructions. Bead-labeled cells were washed in PBS with 0.5% BSA (Roche), washed, and submitted to the UCSF Institute for Human Genetics for single cell RNA sequencing using 10x Genomics Chromium technology. Libraries were prepared by UCSF Institute for Human Genetics, according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics)