thCHART of roX2 lncRNA [DNA]

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 lncRNAs on chromatin. Target RNA is enriched from formaldehyde chromatin extract using biotinylated antisense oligonucleotide. Our new oligonucleotide strategy includes incorporating LNA nucleotides and toehold design to increase specificity and affinity of hybridization. RNA complexes are rinsed under stringent conditions and eluted using elution oligonucleotide.