Multiomics analysis on polyamine metabolisms in colorectal cancer indicates important contribution of impaired anti-tumor activities of CXCR6+CD8+T cells by the extracellular putrescine

The models were reconstructed to randomly obtain three colons in each group. Lamina propria mononuclear cells (LPMC) were extracted as previously described (Wei et al. 2024). Whole cells from the colons were isolated using the RWD Intestine Enzymolysis Kit (DHIE-5007). The LPMC and whole cells with number ratio 1:1 were pooled into single-cell suspension for downstream construction of scRNA-seq libraries using a SeekOne® Digital Droplet single-cell 3’ transcriptome kit (SeekOne, Beijing) according to the manufacturer’s instructions. The scRNA-seq libraries were sequenced at Novogene (Co. Ltd., Beijing, China). The processing of the raw reads followed the same protocol as described previously (Shi et al. 2024). The downstream analysis in R software, including the quality filter, cluster annotation, and DEGs, was the same as mentioned above.

本研究对实验模型进行重建，每组随机获取3段结肠组织。固有层单核细胞（Lamina propria mononuclear cells, LPMC）的提取方法参照既往研究（Wei等，2024）。采用RWD肠道酶解试剂盒（RWD Intestine Enzymolysis Kit，货号DHIE-5007）分离结肠组织全细胞。将固有层单核细胞与全细胞按1:1的细胞数比例混合制备单细胞悬液，随后依照产品说明书，使用SeekOne® 微滴式单细胞3'转录组试剂盒（SeekOne，北京）构建单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）文库用于后续实验。单细胞RNA测序文库交由北京诺禾致源科技股份有限公司（Novogene Co. Ltd., Beijing, China）完成测序。原始测序读段的处理流程参照既往研究（Shi等，2024）中的方案。后续采用R软件开展数据分析，包括质量过滤、细胞聚类注释以及差异表达基因（differentially expressed genes, DEGs）分析，分析流程与前述一致。