GSM9005580: 2312041_E_0116,the naphthalene-induce airway epithelial injury prior to infection with H37Rv group; Mus musculus; RNA-Seq
After sampling, tissue was washed in pre-chilled PBS and dissociated using the Tissue Dissociation Kit A Pro (K01801-30, SeekGene BioTech Co., Ltd., Shanghai, China). Red blood cells were removed using Red Blood Cell Lysis Buffer (R1010, Solarbio), followed by AO/PI staining. Cell quality was assessed using a fluorescence cell analyzer (Countstar®Rigel S2). Based on cell viability and nuclear integrity, dead cells (130-090-101, Miltenyi Biotec) and debris (130-109-398, Miltenyi) were removed. The cells were washed twice in RPMI1640 (11875119, Gibco) and resuspended in RPMI1640 with 2% FBS at a concentration of 1 × 10⁶ cells/mL. scRNA-seq libraries were prepared using the SeekOne® Digital Droplet Single Cell 3' Library Preparation Kit (No.K00202, SeekGene). Cells were mixed with reverse transcription reagents and loaded into sample wells of the SeekOne® chip. Barcoded Hydrogel Beads (BHB) and oil phase were then added to form emulsion droplets. Reverse transcription was performed at 42°C for 90 minutes, followed by 5 minutes of inactivation at 85°C. After emulsion breaking, cDNA was purified and PCR amplified. The amplified cDNA products were fragmented, end-repaired, and ligated with sequencing adapters, followed by purification. The library was purified using Vazyme N411-01 DNA purification beads and analyzed with Qubit (Q33226, Thermo Fisher Scientific) and a Bioptic Qsep400 Fragment Analyzer. Finally, libraries were sequenced on an Illumina NovaSeq 6000 platform with PE150 chemistry, and the raw data was saved in FASTQ format.