GSM8863171: Mouse lung tissue, PBS, 48hr, LDRT 24hr; Mus musculus; RNA-Seq
Mouse lung tissues were isolated and digested using a gentleMACSTM Dissociator and MACS Lung Dissociation Kit (Miltenyi Biotec, Gladbach, Germany). To separate mononuclear cells, the dissociated lung cells were slowly added to Ficoll-Paque™ PREMIUM 1.084 sterile solution (Cytiva, Wilmington, DE, USA) (Ficoll: cells = 3:4 mL). The solution was centrifuged at 400× g for 30 min (20 ℃, decel = 0), the intermediate zone (layer between Ficoll and plasma) was collected and DMEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) was added. The solution was centrifuged at 400× g for 5 min (25 ℃, decel = 9), the supernatant was removed, and the cells were resuspended in PBS. Mouse cell samples were prepared for scRNA-seq using the Chromium Single-Cell 3′ Reagent version 3.1 kit (PN 1000121) and Chromium Single-Cell Controller (10× Genomics, Pleasanton, CA, USA). Subsequently, the cells were loaded onto a Single-Cell G Chip (PN 1000127) for gel bead-in-emulsion generation and barcoding, targeting a recovery of 10,000 cells per sample. Reverse transcription, clean-up, and cDNA amplification were performed to isolate and amplify cDNA or a ligated sequencing adaptor for Illumina library construction, according to the manufacturer's protocol. The libraries were constructed using the Chromium Single-Cell 3′ reagent kit v3.1 (PN 1000121), 3′ Library Construction Kit (PN-1000157) according to the manufacturer's protocol. For sequencing, the library concentration was assessed using a Qubit 2.0 Fluorometer and dsDNA HS Assay Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Single-Cell 3′ gene expression libraries were run on an Illumina NovaSeq 6000 system with 2×100 bp read length.