2.2. RNA Sequencing of Blood Samples
For bulk mRNA sequencing, blood samples were collected at baseline (cycle 1 day 1, C1D1), 24 h after the first mitazalimab administration (cycle 1 day 2, C1D2), and before starting the third treatment cycle (cycle 3 day 1, C3D1). RNA was extracted, processed to reduce globin RNA, and prepared for sequencing using an Illumina TruSeq Stranded mRNA sample preparation kit. Samples were sequenced using a 2 × 50 base pair paired-end sequencing protocol, aligned to references genome using STAR (v2.4), and quantified using RSEM (v1.2.14). The data were further analyzed in R (v4.2.2) and R Studio (v 2023.06.0+421). R scripts and data to reproduce the data analysis are available in a repository on GitHub upon request.Gene counts matrix was analyzed to remove low expressing genes and for samples with large inter-individual Z-score distribution. The 1500 most variable genes based on standardized variance were selected for principal component analysis (PCA). The effects of confounding variables were assessed by calculating the correlation between principal components (PCs) and metadata variables from the study, using the DEGreport package (v1.34) [Heatmaps of gene expression were generated from the Z-scores of VST gene counts using ComplexHeatmap (v2.14) [