Single cell RNA and TCR sequencing of leukocytes in melanoma tumor and draining lymph node (dLN) from mice fed with SF-NIH31 or SF-AIN93G diet
Single cell RNA and TCR sequencing of leukocytes in melanoma tumor and draining lymph node (dLN) from mice fed with SF-NIH31 or SF-AIN93G diet
Mice were fed with SF-AIN93G(SpecialtyFeed) or SF-NIH31 (SpecialtyFeed) for four weeks, followed by subcultaneous injection of 1*10^5 B16-OVA cells. At day 18 post implantation, CD45+ cells were sorted from processed tumours using FACS. scRNA-seq and scTCR-seq libraries were prepared using the SeekOne® DD Single Cell 5′ Transcriptome-seq Kit or Single Cell BCR/TCR Enrichment Kit for mouse T cells (v1.3 Chemistry), following the manufacturer's protocol. Briefly, FACS-sorted cells were washed twice with PBS containing 2% FBS, and approximately 20,000 cells were loaded into the SeekOne® Digital Droplet System for single-cell encapsulation and barcoding. After droplet breakage, cDNA was purified using magnetic cleanup beads and amplified by PCR (98 °C for 3 min; 13 cycles of 98 °C for 20 s, 63 °C for 30 s, 72 °C for 1 min; final extension at 72 °C for 5 min). For gene expression library construction, 50 ng of amplified cDNA was fragmented, end-repaired, adaptor ligated, and subjected to double-sided size selection using SPRIselect beads. Libraries were then indexed via PCR (98 °C for 3 min; 13 cycles of 98 °C for 20 s, 54 °C for 30 s, 72 °C for 20 s; final extension at 72 °C for 5 min), followed by a second double-sided SPRIselect cleanup. Library quality was assessed using an Agilent TapeStation, and sequencing was performed on a DNBSEQ-G400 platform using following configuration: 29 bp Read1, 8 bp N5 Index, 8 bp N7 Index, 90 bp Read2. For scTCR libraries, 3 μL of cDNA post amplification and purification was used for targeted TCR enrichment. The first enrichment was performed using mouse TCR primers 1, followed by SPRIselect bead cleanup. A second enrichment step with mouse TCR primers 2 was conducted, followed by double-sided size selection using SPRIselect beads. Library quality was assessed using an Agilent TapeStation. Enriched products underwent fragmentation, end-repair, adapter ligation, and further SPRIselect bead purification. Libraries were then indexed with adapter-specific primers, and final quality control was performed using an Agilent TapeStation.