Gene expression signature of resident cDCs in tumor draining LNs
RNA-seq and analysis were performed at Rhelixa. In brief, full-length cDNA was prepared from total RNA by SMART-SeqÂ® v4 UltraÂ® 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 Ã? 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).