title: GSM8859536: Caudal lung cells, Ctsl treated, biol rep 2; Mesocricetus auratus; RNA-Seq
library_name: GSM8859536
library_construction_protocol: The caudal lung lobe was excised and preserved in a storage medium containing 1% BSA and 2 μg/mL Actinomycin D (ThermoFisher) in 1xPBS until further processing. The tissue was then transferred to a digestion medium supplemented with 2 μg/mL Actinomycin D, 750 Mandl units/mL Collagenase CLS II (Sigma), and 1 mg/mL DNase I (Millipore Sigma) in 2 mL Dispase medium (Corning). Mechanical dissociation was performed using tweezers for 2 minutes, followed by incubation at 37°C with 5% CO2 for 30 minutes. After digestion, the cell suspensions were filtered through 70 μm syringe strainers (pluriSelect). Red blood cells were lysed using a lysis buffer (BioLegend), and the remaining cells were washed with 0.04% BSA in 1xPBS, resuspended in the same buffer, and filtered through a 40 μm cell strainer (Fisher Scientific) to remove cell clumps before cryopreservation. Upon thawing, cells were filtered again using a 40 μm Flowmi cell strainer (SP Bel-Art). Dead cells were removed with a dead cell removal kit (Miltenyi Biotec), and cell viability was assessed using Acridine Orange/Propidium Iodide (AOPI) staining. The library was prepared using Chromium GEM-X Single Cell 3' Reagent Kits V4, following the manufacturer's instructions.