Role of hNABP2 (hSSB1) in transcriptional regulation

Use Global Run-On assay (GRO)-seq, chromatin immunoprecipitation (ChIP)-seq, and DNA: RNA immunoprecipitation (DRIP)-seq to investigate whether and how hNABP2 (hSSB1) regualte transcription. Overall design: Global Run-On assay (GRO)-Seq: nascent RNA was pulled down by anti-BrDU conjugated beads, the RNA extracted from the GRO reaction was converted into cDNA, and library was prepared using the NEBNext Ultra II directional library prep kit (E7760, NEB). ChIP-Seq: chromatin was crosslinked with formaldehyde, DNA was sheared by sonication, and hNABP2-bound DNA was pulled down by hNABP2 antibod. the ChIPed DNA was end repaired using NEBNext Ultra II End Prep buffer and Enzyme mix, adaptor-ligated, cleaned with 0.9× AMPure beads. The adaptor ligated DNA was then amplified using four (Index 17, 24, 26 and 28) NEBNext Multiplex Oligos for Illumina and universal primers (E7335, NEB) for the two independent experiments. DNA: RNA immunoprecipitation (DRIP)-Seq: The DNA:RNA immunoprecipitation was performed DNA was incubated with S9.6 antibody The RNA was extracted from the DRIP samples using Monarch RNA extraction kit (T2010, NEB). The libraries were constructed using directional RNA library prep kit.