Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation

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Abstract Whereas techniques to map chromatin-bound proteins are well-developed, mapping chromatin-associated RNAs remains a challenge. Here we describe Reverse Transcribe & Tagment (RT&Tag), in which RNAs associated with a chromatin epitope are targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates RNA/cDNA hybrids that are subsequently tag-mented for sequencing by Tn5. We demonstrate the utility of RT&Tag in Drosophila cells for capturing the noncoding RNA roX2 with the dosage compensation complex and maturing transcripts associated with silencing histone modifications. We also show that RT&Tag can detect N6-methyladenosine (m6A)-modified mRNAs, and show that genes producing methylated transcripts are characterized by extensive promoter pausing of RNA polymerase II. The high efficiency of in situ antibody tethering and tagmentation makes RT&Tag especially suitable for rapid low-cost profiling of chromatin-associated RNAs from small samples.

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