Next Generation Sequencing Facilitates Quantitative Analysis of Wild Type and Adar1 B Cell Conditional Knockout Activated B Cells Transcriptomes

Purpose: to compare the gene expression differences between wild-type activated B cells and Adar1 knockout activated B cells Methods: In vitro activated B cells were sorted using a BD FACSAira sorter, and total RNA 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). Overall design: Mouse splenic B cells from wild-type and Adar1 B cell conditional knockout mice were purified using anti-CD43 microbeads and cultured 36 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, 1 µg/ml anti-CD40 and 10 µg/ml anti-IgM at 37? with 5% CO2.