GSM8486402: PBS treated rep2, scRNA; Mus musculus; RNA-Seq
Tumor samples treated with CAR MEV or PBS were collected in RPMI 1640 medium and rapidly processed to minimize ischemic time. The tissues were washed with 1× DPBS, cut into 2-4 mm³ pieces, and dissociated in an enzyme mixture using the gentleMACS Octo Dissociator at 37°C for 30 minutes. After centrifugation, the cells were resuspended in RPMI 1640 or DMEM, filtered through a 70 µm strainer, and subjected to red blood cell lysis. The cell concentration was adjusted to 700-1200 cells/µl and immediately processed for 10x Genomics® Single Cell sequencing. Single-cell RNA sequencing libraries were generated using the 10X Genomics Chromium NextGEM Single Cell 3' Kit v3.1 according to the manufacturer's protocol. Briefly, single-cell suspensions were prepared and mixed with RT reagents, including template switch oligos and enzymes, and loaded onto Chromium NextGEM Chip G. The chips were processed using the 10X Genomics Chromium Controller to generate Gel Beads-in-Emulsions (GEMS). Reverse transcription (RT) was performed within the GEMS, followed by cDNA amplification.The cDNA was purified using SPRIselect beads and subsequently fragmented, end-repaired, and A-tailed. Adapter ligation was carried out, and the libraries were enriched by PCR. Post-PCR, the libraries were purified and size-selected using SPRIselect beads to ensure the appropriate fragment size distribution.The final libraries were quantified using a Qubit fluorometer and assessed for quality using an Agilent Bioanalyzer. Sequencing was performed on an Illumina NovaSeq X Plus platform in paired-end 150 bp mode.