Potent anti-tumor immunity and reversal of CD8 T cell exhaustion by spatially and functionally targeting Treg cells in the tumor microenvironment

1×10⁶ of splenic GFP⁺ Tregs were purified from WT (n=4) and KO mice (n=4) via FACS isolation. RNA was extracted using RNeasy Micro Kit (Qiagen) following its standard protocol and RNA degradation and contamination was monitored on 1% agarose gels. For RNA sequencing, libraries were prepared using NEBNext® Ultra TM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated. All samples were prepared at the same time and sequenced on the same lane. Original image data file from high-throughput sequencing platforms (like Illumina) is transformed to sequenced reads (called Raw Data or Raw Reads) by CASAVA base recognition (Base Calling).  Raw data in FASTQ format were processed through fastp. In this step, clean data (clean reads) were obtained by removing reads containing adapter and poly-N sequences and reads with low quality from raw data. All the downstream analyses were based on the clean data with high quality. Reference mouse genome and gene model annotation files were downloaded from genome website browser (NCBI/UCSC/Ensembl). Paired-end clean reads were mapped against the reference genome using the Spliced Transcripts Alignment to a Reference (STAR) software. FeatureCounts was used to count the read numbers mapped of each gene. And then RPKM (Reads Per Kilobase of exon model per Million mapped reads) of each gene was calculated based on the length of the gene and reads count mapped to this gene. The FPKM gene expression matrix, R code, DEG list, and targeted gene list are uploaded in this project.