GSM9045743: TCR-seq, B16-F10 melanoma cells, scrambled control (SCR) , rep2; Mus musculus; OTHER
For Ribo-Seq, cells were incubated with 100 μg/ml Cycloheximide (MCE) for 8 min at 37°C and were then lysed on ice for 10min in 400uL lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/ml Cycloheximide, 1% Triton X-100). The supernatant was collected via centrifugation at 12 000 g for 10 min at 4 °C, and its' absorbance at 260nm was measured. The ribosomes with equal A260 in each group were reserved for RNA sequencing. For TCR-Seq, PBMCs were isolated from whole blood of C57BL/6N mice that had been inoculated with either control B16F10 cells or Mettl5-KO cells 12 days prior.   For scRNA-Seq,  Tumors were surgically resected and preserved in MACS Tissue Storage Solution, then transferred to the laboratory at low temperature. Following the manufacturer's instructions for the Miltenyi Biotec dissociation kit, the tumors were dissociated into a single-cell suspension. Post-dissociation, cells were resuspended in MACS Tissue Storage Solution to maintain viability. Cell counting and concentration adjustment were performed, followed by labeling with Zombie NIR viability dye to distinguish live and dead cells. Subsequently, cells were incubated with Fc blocker in FACS buffer to prevent non-specific antibody binding. Cells were then incubated with an antibody cocktail containing anti-CD45 antibodies. After incubation, cells were washed, filtered through a 70-μm Macs SmartStrainer to remove aggregates and debris, ensuring a high-quality single-cell suspension. Finally, CD45+ live immune cells were sorted using a BD FACSAria II instrument for downstream scRNA-seq analysis with the 10X Genomics 5' platform. This method ensures the isolation of viable immune cells, crucial for studying the tumor immune microenvironment. For Ribo-Seq, added 15U RNase1 (Thermo Fisher Scientific) for each 1OD of lysate, and incubated the reactions at 22°C for 40 min with gentle mixing. Ribosome-protected fragments (RPFs) were collected by MicroSpin S-400 columns (Cytiva) and purified by Zymo Research RNA Clean & Concentrator-25 kit (Zymo). The RPFs were then separated with 15% polyacrylamide TBE–urea gel, excised the gel slices between 17 and 34 nucleotides and recovered RNA from PAGE gel by ZR small-RNA™ PAGE Recovery Kit (Zymo). Purified RNA was treated with PNK(NEB), and the RNA library was prepared by VAHTSTM Small RNA Library Prep Kit for Illumina®(Vazyme). The library concentration was analyzed using the VAHTSTM Library Quantification Kit for Illumina®, and equimolar pools of libraries were sequenced. For TCR-Seq, DNA was extracted from PBMC using a QIAamp DNA Blood Mini Kit (Qiagen), and the concentration was tested using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The DNA was used as a template for PCR amplification, which was performed to generate the library of TCR and BCR. The step 1 PCR amplification protocol was as follows: 95 °C for 5 min; 95 °C for 30 s, 59 °C for 30 s; 72 °C for 1 min for 30 cycles; and 72 °C for 10 min. The step 2 PCR amplification protocol was as follows: 98 °C for 2 min; 98 °C for 30 s, 65 °C for 30 s; 72 °C for 30 s for 10 cycles; and 72 °C for 5 min. The PCR products were purified, and the barcodes were confirmed. The annealed region of these primers was the CDR3 region, as shown by Multiplex PCR Amplifies. The CDR3 regions were sequenced on Illumina NextSeq500 (MyGenostics).  For scRNA-Seq, Following cell sorting, live CD45+ immune cells were resuspended in PBS + 0.04% BSA at 1,000 cells/μL. Libraries were prepared using the 10x Genomics Chromium Next GEM Single Cell 5' v2 Kit according to manufacturer protocol, targeting 10,000 cells per sample. cDNA amplification and fragmentation were performed before Illumina adapter ligation.