GSM7596253: Adult murine lung immune cells,replicate 1, scRNA-seq; Mus musculus; RNA-Seq
Left lung lobes from wild type adult mice infected influenza PR8 14 days were digested by gentle shaking at 37℃ in lysis buffer containing Dispase II and Collagenase type I. Tissue were dissociated to single cells and the following antibodies were used to sort immune cells: Rat anti-FITC CD45, Rat anti-FITC CD31, Rat anti- APC Epcam.  The cells were subjected to fluorescence activated cell sorting (FACS) to select all Epcam-CD31-CD45+cells. Sorted dissociated cells were washed with 1× DPBS containing 2% FBS. Cells were stained with AO/PI, and viability was assessed using a Countstar     Fluorescence Cell Analyzer (Aber Instruments). Exposure cells to lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA  synthesis, each cDNA molecule was tagged on the 5'end (that is, the 3'end of a messenger RNA transcript) with UMI and cell label indicating its cell of origin. Briefly, 10× beads that were then subject to second-strand cDNA synthesis, adaptor ligation, and universal amplification.Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3' end of the transcripts linked with the cell barcode and UMI. All the remaining procedures including the library construction were performed according to the standard manufacturer's protocol (Chromium Single Cell 3ʹ v3. 1). Sequencing libraries were quantified using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo  Fisher Scientific). The libraries were sequenced on NovaSeq6000 (Illumina), and 150 bp paired-end reads were generated. The sequencing was performed by Majorbio Co., Ltd (Shanghai, China).