Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Wac B Cell Conditional Knockout Activated B Cells Transcriptomes

To investigate the function of Wac in the regulation of B cell activation and plasmablasts differentiation, we used Cre-Loxp system to generate Wac conditional knockout mice (Wacf/f Cd19cre/+). We then performed gene expression profiling analysis using data obtained from RNA-seq of in vitro LPS activated wild-type and Wac knockout B cells. Overall design: Splenic B cells from wild-type and Wac Cd19-cre conditional knockout mice were purified using anti-CD43 microbeads and cultured 24 hours at the concentration of 2 million cells per ml in DMEM medium supplemented with 10% FBS, 1% penicillin/streptomycin, 1% non-essential amino acids and 20 µg/ml LPS at 37? with 5% CO2. Total RNA of in vitro activated B cells was extracted using RNeasy Mini Kit (Qiagen) and poly(A) mRNA was isolated with the poly(A) mRNA Magnetic Isolation Module (NEB). Libraries were prepared using NEBNext Ultra RNA Library Prep Kit for Illumina (NEB) and sequenced on an Illumina Hiseq instrument (Illumina). Sequence reads were filtrated using Cutadapt (V1.9.1), and then mapped to the reference genome sequences (GRCm38.98) using Hisat2 (V2.0.1). Gene expression level was calculated using Htseq software (V0.6.1).