ChIP of H4K16ac

Understanding the targeting and spreading patterns of lncRNAs on chromatin requires a technique that can detect both high intensity binding sites and reveal genome-wide spreading patterns with high confidence. We developed an improved hybridization capture protocol to determine lncRNA localization using biotinylated LNA-containing oligonucleotides that hybridize to the target RNA and enhance capture specificity by including a protecting oligonucleotide that competitively displaces contaminating species, leading to highly specific RNA capture. This approach revealed the spreading pattern of roX2, a lncRNA involved in dosage compensation in D. melanogaster, how this pattern relates to chromatin features, and how spreading of roX2 changes upon cellular stress.  Upon heat shock, roX2 displays reduced spreading on X chromosome and surprising relocalization to sites on autosomes, revealing how this improved hybridization capture approach can reveal previously uncharacterized changes in the targeting and spreading of a lncRNAs on chromatin. Experiment contain 2 replicates of control, heat shock and heat shock + recovery samples from S2 cells.