ChIP-seq and RNA-seq of Transformed B Cells

Cells were prepared for ChIP-seq or ChIP-qPCR analysis according to Yamane et al. (2013) (link) with minor modifications. For each experiment, harvested cells were depleted of dead/apoptotic cells using the dead cell removal kit (Miltenyi Biotec) before further processing. For CEBPA experiments, myeloid cells were additionally enriched using anti-CD11B micro-beads (Miltenyi). Primary transformed and untransformed B cell precursors were typically harvested on day 7 after transduction; for CEBPA experiments, cells were harvested after 7 days of DOX addition. For library preparation, the NEBNext ChIP-Seq Library Prep Master Mix Set and NEBNext Multiplex Oligos for Illumina (NEB) were used. For RNA-seq, cells were isolated, cultured, and harvested as for ChIP-seq experiments. RNA was extracted in duplicate experiments from 5 × 10⁶ cells using the QIAGEN RNeasy mini kit. Libraries were generated from pre-enriched mRNA using the NEBNext mRNA Library PrepReagent Set for Illumina (NEB) and pre-amplified using the NEBNext Multiplex Oligos for Illumina (NEB). A detailed description of the experimental procedures, bioinformatic analyses, and statistics is available in the Supplemental Experimental Procedures.