GSM8657655: FLT3L 2; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane and euthanized by intra-cardiac PBS/heparin perfusion. Tissues were harvested, minced, and digested in HBSS (Gibco) supplemented with 2mg/mL collagenase-A (Roche), and 50μg/mL DNase-I (Sigma), for 30min at 37°C with constant agitation. Tissue digestion was neutralized with 10% FBS (R&D) and digests were filtered through 40μm cell strainers, then cells were counted with a Countessa-II automated cell counter (Life technologies). Samples were then stained with fluorescently conjugated anti-CD45 antibody, washed, and resuspended in FACs buffer containing 7-AAD (BD Biosciences) to distinguish live cells. Cell suspensions were then sorted for viable immune cells (7-AAD-/CD45+) using an Aria-II cell sorter (BD Biosciences). Cell sorting was done through an 85um nozzle into ice-cold PBS supplemented with 20% FBS. Sorted cells were washed and resuspended in PBS with 0.04% BSA. Cell concentration and post-sort viability were measured with a BD Rhapsody single cell system (BD Biosciences). Libraries were sequenced on a NovaSeq 6000 sequencing system (Illumina) with a minimum target of 50,000 mean reads per cell. The cellranger mkfastq pipeline (10X Genomics) was used to demultiplex Illumina base call files to FASTQ files.