RNA-seq analysis of follicular (FO) and marginal zone (MZ) B cells, isolated from TLR9WT or TLR9-/- BALB/c mice, stimulated with TLR7 agonists (CL097, Invivogen) or left unstimulated .

Purpose: The goal of this study was to assess if increased TLR7 signaling could be seen in TLR9-/- compared to TLR9WT FO and MZ B cells. Methods : FO and MZ B cells from TLR9WT or TLR9-/- BALB/c mice were sorted using FACSaria. A minimum of 100,000 cells per B cell subsets were sorted. Sorted B cell subsets were stimulated for 4 hours at 0.25M/ml with TLR7 agonist CL097 (Invivogen) at 5µg/ml or left unstimulated. RNA was isolated using the RNeasy Plus Micro Kit (QIAGEN). Samples were sequenced on an Illumina Next-seq 2000 using a P3 200 cycle flowcell (Illumina, Inc) with 2 x 101 bp paired-end reads (20 million reads per sample). Results: There were no significant DEG (FDR > 0.05) following TLR7 stimulation between TLR9WT and TLR9-/- FO or MZ B cells. mRNA profiles of TLR9 deficient (TLR9-/-) and intact TLR9 (WT) FO and MZ B cells of BALB/c mice, stimulated with TLR7 agonists (CL097) or unstimulated, were generated in three replicates by sequencing, on an Illumina Next-seq 2000 using a P3 200 cycle flowcell.