GSM7886200: BALC-7d-17; Sus scrofa; RNA-Seq
After euthanasia, the lungs were aseptically extracted with clamping the tracheal tube, and gross lesions were measured. The left mainstream bronchus was clamped, and tissue samples were collected from the clamped left lung lobes for RNA extraction and histopathological examinations. The right lung was lavaged with 15 mL of sterile PBS, containing 100 μg/mL ampicillin (USB Corporation Cleveland, OH, USA) and Anti-Anti, to retrieve the first lavage fluid. The second lavage was subsequently conducted with 75mL of PBS to collect the second lavage fluid. The collected lavage fluid was centrifuged at 1000 × g in room temperature for 10 min to separate the bronchoalveolar lavage fluid (BALF) and cells (BALF cells). The resulting supernatant or BALF from the first lavage were used for quantifying the protein levels of cytokine/chemokine. The cell pellet or BALF cells from both the first and second lavage were merged and used for flowcytometry, quantification of mRNA expression levels, and single cell RNA sequencing. The BALF cells were washed three times with PBS after red blood cell (RBC) lysis. The tissues and BALF as well as BALF cells were collected in tubes, snap-frozen using liquid nitrogen and stored immediately at -80°C for further analysis. For scRNA-seq library construction, BALF cells were washed with cold Ca2+ and Mg2+ free 0.04 % BSA/PBS at 300 g for 5 min at 4°C. Samples were gently resuspended in cold Ca2+ and Mg2+ free 0.04% BSA/PBS, and then counted with a LUNA-FX7™ Automated Fluorescence Cell Counter (Logos Biosystems, Anyang, Gyeonggi-do, Republic of Korea) using acridine orange and propidium iodide staining. ScRNA-seq libraries were constructed with the Chromium Single Cell 3' v3.1 assay (10x Genomics, Pleasanton, CA, USA) using 36 BALF cells. According to the protocol, libraries were prepared using the 10x Chromium Controller and Next Gem Single cell 3' Reagent v3.1 kits. Briefly, the cell suspension (target recovery 10,000 cells) was mixed with reverse transcription master mix and loaded with Single Cell 3′ Gel Beads and Partitioning Oil into a Single Cell G Chip to generate single-cell Gel Bead-in-emulsion (GEM). Poly-adenylated mRNA from single cells were uniquely barcoded and reverse-transcribed within GEM. After barcoded full-length cDNA was generated from mRNA through GEM-RT incubation, the barcoded cDNA molecules were amplified with PCR. For 3' Gene Expression Library construction, the enriched cDNA was sequentially subjected to the enzymatic fragmentation, end-repair, A-tailing, adaptor ligation and index PCR. The purified libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide and qualified using the Agilent Technologies 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA). Then, the libraries were sequenced using NovaSeq 6000 System (Illumina, Inc., San Diego, CA, USA), and 150 bp paired-end reads were generated. The sequencing depth of 3' Gene Expression Library was about 20,000 read pairs per cell.