Rb1+/+ versus Rb1?S/?S RNA Seq

Upon G1-S transition, cyclin-dependent kinases (CDKs) phosphorylate the retinoblastoma tumor suppressor protein (pRB) to release E2F transcription factors, which activate transcriptional programs, required for S-phase entry. Beyond the G1-S transition, pRB activity remains poorly understood. Our lab has discovered that hyperphosphorylated pRB (ppRB), found beyond G1, retains exclusive binding to E2F1 through an alternate E2F1-‘specific’ binding site at the pRB c-terminus. We have developed a gene-targeted mouse model that is defective for the E2F1-‘specific’ interaction. We are exploring the function of this complex through genome-wide expression profiling. Overall, this work suggests an alternate pRB-E2F1 complex persists beyond the G1-S transition to establish regions of constitutive heterochromatin. Overall design: 1. Total RNA from passage 4 quiescent MEFs isolated using TRIzol RNA extraction protocol 2. rRNA was depleted from total RNA using the RiboMinus Euk System V2 protocol according to manufacturer’s procotol 3. rRNA-depleted RNA samples were submitted for picoanalyzer analysis to determine concentration, purity, and rRNA content 4. Three wild-type and three mutant RNA samples with <10% rRNA remaining were submitted for library construction 5. Library was used for Illumina HiSeq 2500 paired end sequencing.