Delineating functional enhancers (cis-regulome) in LPS-activated murine splenic B cells

LPS activated murine splenic B cells (aB) were subjected to FAIRE-Seq, STARR-Seq, Hi-C, ChIP-seq, Nascent-RNA-seq and RNAseq to compile cis-regulome. Splenic B cells were isolated from C56BL/6J mice (6-8 weeks of age) using B cell isolation kit from Miltenyi (Catalog # 130-090-862) and cultured in complete RPMI with 10% FCS. Purified B cells (250,000 cells/ml) were activated with LPS (10ug/ml, Salmonella typhimurium, Sigma Catalog # L6143). At 72h post-activation B cells were subjected to FAIRE-Seq, Hi-C, ChIP-seq, Nascent-RNA-seq (EU pulse-labeling) and RNAseq. STARR-Seq was performed by transfecting FAIRE-Seq plasmid library at 48h or 60h post LPS-activation. RNA was isolated at 72h post-activation and processed for STARR-Seq. STARR-Seq_24h refers to transfection of library at 48h and STARR-Seq_12h refers to transcfection at 60h. By computational integration of FAIRE-seq and STARR-seq datasets (HOMER peak calls) we identified approximately 10,000 functional enhancers in LPS-activated B cells. These enhancers were connected to active promoters identified by RNA-seq using Hi-C. Integrated datsets yielded a functional mammalian cis-regulome for activated B cells. Cis-regulome was further analyzed using ChIP-seq for 3 histone marks H3K4me1, H3K4me3 and H3K27ac. Nascent RNA-Seq was used for localizing eRNAs and determining rates of gene transcription. Resting B-cells (rB) were used as controls.