title: single cell RNA sequencing of lung in germ-free and specific pathogen free mice
design: single cell RNA sequencing of lung in germ-free and specific pathogen free mice
library_construction_protocol: Animals were sacrificed using injection of pentobarbital and the lung was perfused with cold PBS. Subsequently a catheter was inserted into the trachea and 1 ml of 5 mg/ml Dispase solution was injected. The solution was incubated for 5 min in the lungs before it was excised and placed in IMDM on ice. The lung was then minced and incubated with an enzyme mix containing 1 mg/ml Dnase I, 2 mg/ml Collagenase IV and 5mg/ml Dispase for 20 min at 37 °C, shaking. The lung was then transferred to a 70 μm cell strainer and smashed through with 20 ml Ca/Mg-free PBS to get a single cell suspension. The cells were then collected at 4 °C for 10 min at 500g. The pellets were resuspended in 1ml ACK buffer (Gibco, A1049201) and incubated for 1 min at room temperature. Then 14 ml of Ca/Mg-free PBS was added, and cells were centrifuged at 500g for 7 min. The cells were then incubated for one hour on ice in 500 μl Miltenyi Dead Cell Removal kit beads, after which 5.5 ml of kit Binding Buffer was added to each sample. The samples were filtered and then loaded onto LS-columns and the flow-through was collected. The flow through was then centrifuged 7 min 500g and was resuspended in Ca/Mg-free PBS with 0.04% BSA and samples proceeded directly to scRNAseq.  according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide according to 10x genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 User Guide