title: GSM8504079: Con-2; Mus musculus; RNA-Seq
library_construction_protocol: Fresh lung tissues were resected from mice and stored in tissue preservation solution (Shanghai Biotechnology Corporation, 21903-10). Take the tissue into a sterile culture dish, add 5 mL of 1 X DPBS precooled at 4 ℃ for rinsing, repeat 1-2 times to remove residual tissue protective solution. Place the moistened tissue in a new 1.5 mL EP tube, add a small amount of digestive solution (500 μL), and use clean scissors to cut the tissue as much as possible. Transfer the shredded tissue to a 50 mL centrifuge tube containing 5 mL of digestive solution, and place it in a 37 ℃ water bath for shaking digestion. Gently shake the digestive solution well, do not shake vigorously. Collect the first sample after about 15 minutes of digestion, add enzyme solution again and digest for 20 minutes before collecting for the second time (if there are fewer cells and residual tissue after the first digestion, second digestion can be used). Pass the cell suspension collected twice through prewetted 40 μm filter membrane. Collect the filtrate into a new 50 mL centrifuge tube through M-cell sieve. Centrifuge all collected cell suspensions at 2000 rpm and room temperature for 10 minutes, then discard the supernatant. If there is a red color in the cell precipitate, it indicates that there are red blood cells in the precipitate. Add 2 mL of precooled 1 X red blood cell lysis solution to the cell precipitate, gently blow and mix, do not vortex, and incubate at room temperature for 3 minutes. After the red blood cell split time, centrifuge at 1500 rpm for 5 minutes at room temperature, discard the supernatant, add an appropriate amount of 1 X DPBS (including 2% FBS) to resuspend the cells, mix well, count, and observe the cell state and debris under a microscope Dissociated cells were washed with 1 X DPBS containing 2% FBS. Cells were stained with 0.4% Trypan blue (Thermo Fisher, Cat. no. 14190144) to check the viability on Countess® II Automated Cell Counter (Thermo Fisher). 10× library preparation and sequencing 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 (CG000206 RevD). 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) using 2x150 chemistry.