GSM9107651: SM cells, PBS/NDV-WT/NDV-GP group, lung (merged data); Mus musculus; RNA-Seq
To obtain SM CD4 T cells in lung, lungs were mechanically minced and digested using with 1 mg/mL collagenase I (Sigma, c0130) and mashed through filters. Then, lymphocytes were enriched using Percoll (GE Healthcare, 17-0891-09) density gradient. To obtain SM CD4 T cells in spleen, cells were harvested by mashing the spleens through cell strainer (BD Falcon). Next, lymphocytes were stained with flow cytometry antibodies specicifc for CD4, Va2, CD45.1, and CD45.2 and SM cells were FACS-sorted. The library preparation for single-cell RNA sequencing (scRNA-seq) using the BD Rhapsody platform according to manufacturer instructions. scRNA-seq begins with the capture of individual cells which employs microfluidic technology to isolate cells within nanowells. Once captured, each cell is lysed, and reverse transcription is performed to convert the RNA into complementary DNA (cDNA). During this process, unique molecular identifiers (UMIs) and cell barcodes are incorporated into each cDNA molecule, enabling accurate quantification and differentiation of transcripts from different cells. Following reverse transcription, the cDNA undergoes amplification through PCR to generate an adequate quantity of material for sequencing. The amplified cDNA is then processed to prepare sequencing libraries, which involves enzymatic fragmentation, adapter ligation, and purification steps to ensure that only the desired fragments are included in the final product. To confirm that the libraries meet the necessary standards for sequencing, quality and quantity assessments are conducted using methods such as bioanalyzer or Qubit. Finally, the libraries were sequenced using paired-end sequencing, with 150 nucleotides on each end, conducted in a single lane of a NovaSeq 6000 for each sample. Raw gene expression matrices were generated using the Cell Ranger pipeline (version 6.1.1), coupled with the mm10 genome annotation reference.