ChIP-seq Profiling of Transcription Factor Binding

Chromatin was prepared from pellets of 1 × 10⁶ transduced, cultured B cells as described in (12 (link)). Cross-linking was performed using 1% formaldehyde (Millipore-Sigma, Darmstadt, Germany) and halted using glycine. Pellets were flash-frozen in liquid nitrogen prior to sonication. Thawed pellets were lysed in lysis buffer supplemented with Halt Protease Inhibitor (ThermoFisher Scientific, Rochester, NY, United States), and sonicated for 25 cycles using the Bioruptor UCD-300 (Diagenode, Sparta, NJ, United States). Immunoprecipitation of FLAG-bound chromatin was performed using anti-FLAG M2 magnetic beads (MilliporeSigma, Darmstadt, Germany). Eluted DNA was purified with QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). qPCR on purified DNA was performed as described above, using primers shown in Supplementary Table S1. Threshold cycle values were used to calculate enrichment, represented as percent input. ROIs were identified by analysis of published ChIP-seq data (GEO accession code: GSE58128) (14 (link)). ChIP-seq was performed as described in Solomon et al. (14 (link)). Quality control for chromatin enriched by anti-FLAG antibody was performed by qPCR analysis for association with the IgH intronic enhancer. Sequencing was performed by Genome Quebec on two independent replicates of anti-FLAG ChIP chromatin as well as on input chromatin DNA.