Single-cell RNA Sequencing of CD11c+ Cells

The pooled enriched cells were also used for transcriptome profiling of CD11c + cells. The RNA from enriched cells was isolated using Trizol, and mRNA was enriched using Next Poly (A) mRNA Magnetic Isolation Module (NEB) as per manufacturer’s instructions. One nanogram of purified mRNA then used to prepare cDNA library using PCR-cDNA Sequencing Kit (Oxford Nanopore) as per manufactures’ instructions. RNA sequencing was then performed on the MinION MK1b device (Oxford Nanopore) using FLO-MIN106D Flow cells (Oxford Nanopore).
The output reads were analyzed using Minimap2, Samtools, and Stringtie packages^{18 (link)–20 (link)}. The normalized final counts then were analyzed and visualized using R packages (ggsankey) and Graphpad Prism²¹ .