GSM8242941: Vehicle-OVA; Mus musculus; RNA-Seq
The specific method involves obtaining mouse nasal mucosa and digesting it into a single-cell suspension using hyaluronidase (250U ml-1), collagenase I (114U ml-1), and DNase I (25μg ml-1). Subsequently, CD45+ cells are isolated using Magnetic Activated Cell Sorting (MACS) to prepare a CD45+ single-cell suspension, which is then subjected to single-cell sequencing. 1. The prepared CD45+ single-cell suspension, 10X barcode gel beads, and oil are each added to different chambers of the Chromium Chip M, and GEMs (Gel Beads-in-emulsion) are formed using the 10X Genomics Chromium X system. 2. GEMs are transferred to a PCR machine for reverse transcription, where gel beads containing reverse transcription primers with a 30nt oligo-dT enable the reverse transcription of poly-A RNA in the cells into single-stranded cDNA with Barcode and UMI information. 3. The single-stranded cDNA is purified using magnetic beads. 4. The purified cDNA undergoes PCR amplification. 5. The cDNA concentration is measured using Qubit, and fragment size is assessed using Agilent 2100. 6. After amplification of cDNA, the fragments are enzymatically sheared and size-selected with magnetic beads, followed by end repair, A-tailing, and adaptor ligation with Read2 sequencing primers. A cDNA library with P5 and P7 adaptors is then constructed using PCR. 7. The library is purified using magnetic beads. 8. The library concentration is measured using Qubit, and fragment size is assessed using Agilent 2100.