GSM9018269: Lung cells, APC, biol rep 2; Mus musculus; RNA-Seq
Lung tissues were transported in In sterile culture dish with 10 ml 1x Dulbecco's Phosphate-Buffered Saline (DPBSThermo Fisher, Cat.  no.  14190144) on ice to remove the residual tissue storage solution, then minced on ice.  Weused dissociation enzyme 0.25% Trypsin (Thermo Fisher, Cat.  no.25200-072) and 10 ug/mL 1DNase I (Sigma, Catno. 11284932001) dissolyed in PBS with 5% Fetal Bovine Serum (FBS: Thermo Fisher, Cat.  no.  SV30087.02) todigest the tissues.  Lung tissues were dissociated at 37℃ with a shaking speed of 50 r.p.m for about 40 min. Werepeatedly collected the dissociated cells at interval of 20 min to increase cell vield and viability, Cell suspensions were filtered using a 40 um nylon cell strainer and red blood cells were removed by 1X Red Blood Cell LysisSolution (Thermo Fisher, Cat.  no.  00-4333-57).  Dissociated cells were washed with 1x DPBS containing 2% FBSCells were stained with 0.4% Trypan blue (Thermo Fisher, Cat.  no.  14190144) to check the viability on CountessRI Automated Cell Counter (Thermo Fisher). Library was performed according to the manufacter's instructions (single cell 3' v2protocol, 10x Genomics). Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation, so that each cell was paired with a bead in a Gel Beads-in-emulsion(GEM).  After exposure to cell 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 sequencing were sequenced on Novaseq Xplus using PE150 mode