Fitc anti cd45 30 f11

Tumor single-cell suspensions were prepared from 6 mice inoculated with WT (n = 3) or Hdac3^+/− (n = 3) MCA205 cells for 6 days as described above. Dead cells were removed with vivid yellow staining (Invitrogen, catalog no. L34960). Cells were then stained with FITC anti-CD45 (30-F11; BioLegend, catalog no. 103107), PE anti-CD8a (53-7.6; BioLegend, catalog no. 100707), and APC-Cy7 anti-CD4 (GK1.5; BioLegend, catalog no. 100413) for 30 minutes. Sorting via FACS was performed on a FACS Aria III (BD Biosciences), and 1,000 CD45⁺CD4⁺ or CD45⁺CD8⁺ T cells from every tumor were sorted into separate tubes and resuspended in RNAlater (Thermo Fisher scientific, catalog no. AM7020). Full-length cDNA synthesis and amplification were prepared using SMART-Seq V4 Ultral Low Input RNA Kit (Takara, catalog no. R400752) for sequencing, which is based on the SMART-seq2 method (34 (link)). The Nextera XT DNA Library Preparation Kit (Illumina Inc., catalog no. 15032354) was then used to make cDNA libraries suitable for Illumina sequencing. Briefly, cDNA was fragmented, and an adapter was added using transposase, followed by limited-cycle PCR to enrich and add index to the cDNA fragments. The final library quality was assessed with an Agilent TapeStation. These libraries were sequenced using the Novaseq 6000 system (Illumina Inc.) with 2*150 bp paired-end sequencing.