Bulk RNA-seq of lung-resident memory B cells and comparison to splenic memory and naive B cells

The aim was to utilise bulk RNAseq to compare the transcriptional profiles of memory B cells (Bmem) from the lung tissue to circulatory Bmem derived from spleen, in the context of X31 influenza-induced immune memory. In this study we utilise AID-Cre Rosa-Stop-YFP reporter mice to track previously activated B cell populations, and combine this with an X31-influenza-specific HA probe to isolate flu-specific Bmem. We find that lung Bmem express a number of tissue retention-associated genes (Cd44, Cd69) and downregulate circulatory genes (Sell, S1pr1), in comparison to splenic Bmem. We also identify a number of chemotactic factors (Cxcr3, Ccr1, Ccr5) that are expressed at higher levels in lung Bmem than splenic Bmem. Overall design: Bmem were FACS-sorted from the lungs (IV CD45- B220+ AID-YFP+ GL7- CD69+ infuenza HA+) or spleens (B220+ CD38+ AID-YFP+ GL7- Infuenza HA+) of influenza-infected AID-cre Rosa26-Stop-dTomato mice at day 45 postinfection. These memory populations were isolated the same animals and compared to naïve B cells (Live B220+ CD38+ AID-YFP- GL7- spleen cells) from the same donors. in vivo labelling (IV Cd45) was used in lung samples to allow distinction of Bmem cells in the tissue parenchyma. Lung cells from 6 mice were pooled per biological replicate. Three experiments were performed, each containing a single biological replicate. Technical replicates were acquired in two of these experiments. Each sample has 4x raw fastq files, representing the sample being spread across 4 sequencing lanes