Gene expression signature of resident cDCs in tumor draining LNs. Mus musculus strain:C57BL/6j

RNA-seq and analysis were performed at Rhelixa. In brief, full-length cDNA was prepared from total RNA by SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Clontech). RNA-Seq analysis was performed using the NovaSeq 6000 (Illumina Inc., San Diego, CA) in the paired-end 2 × 100-bp cycle mode with NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs). The quality of the raw paired-end sequence reads was assessed with FastQC (Version 0.11.5; - 10 - https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Low quality (< 20) bases and adapter sequences were trimmed by Trimmomatic software (Version 0.38) with following parameters: ILLUMINACLIP: path/to/adapter.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36. The trimmed reads were aligned to the reference genome using RNA-seq aligner HISAT2 (Version 2.1.0). The HISAT2-resultant .sam files were converted into .bam files with samtools and used to estimate the abundance of uniquely mapped reads with featureCounts (Version 1.6.3). The raw counts were normalized with transcripts per million (TPM).

本研究的RNA测序（RNA-seq）及分析工作均由Rhelixa公司完成。简言之，实验采用Clontech公司的SMART-Seq® v4 Ultra® 低起始量RNA测序试剂盒，从总RNA中制备全长cDNA。RNA测序分析则使用Illumina公司（美国加利福尼亚州圣地亚哥）的NovaSeq 6000测序平台，采用双端2×100bp循环测序模式，并搭配New England Biolabs公司的NEBNext Ultra II RNA建库试剂盒完成文库制备。采用FastQC软件（版本0.11.5；https://www.bioinformatics.babraham.ac.uk/projects/fastqc/）对原始双端测序读段的质量进行评估。使用Trimmomatic软件（版本0.38）对质量值低于20的碱基及接头序列进行截切，具体参数设置如下：ILLUMINACLIP: path/to/adapter.fa:2:30:10 LEADING:20 TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36。使用RNA测序比对工具HISAT2（版本2.1.0）将截切后的读段比对至参考基因组。将HISAT2输出的.sam格式文件通过samtools转换为.bam格式文件，随后采用featureCounts软件（版本1.6.3）计算唯一比对读段的丰度。原始计数采用每百万转录本数（transcripts per million, TPM）进行标准化处理。