Incomplete removal of ribosomal RNA can affect chromatin RNA-seq data analysis

Next-generation sequencing has become one of the major approaches to investigate transcription regulation. RNA-seq, which sequences the RNA complement, can provide a snapshot of the steady-state level of RNA. In addition to whole-cell analysis, RNA-seq can be performed on different cellula r fractions, such as chromatin, nucleoplasm, and cytoplasm, to investigate post-transcriptional regulation, for example. Chromatin RNA-seq provides a picture of the RNAs transcribed by RNA polymerase (pol) I, pol II, and pol III, associated with chromatin, which is a combination of nascent and processed transcripts. As chromatin RNA-seq cannot rely on a poly(A) tail enrichment method, a ribosomal (r)RNA-depletion step is performed during library preparation to avoid an over-representation of rRNAs in the sequencing data. The variety of commercial kits and protocols that are available each has its own strengths and weaknesses [Citation1]. Contrary to the analysis of the data obtained from standard whole-cell RNA-seq or techniques investigating simultaneously nascent transcription of the three polymerases, such as precision nuclear run-on (PRO-seq), no bioinformatics step to remove the remaining rRNA reads is generally performed in chromatin RNA-seq [Citation2–4].