sonication step  (Eppendorf AG)

Journal: Nucleic Acids Research

Article Title: RADIP technology comprehensively identifies H3K27me3-associated RNA–chromatin interactions

doi: 10.1093/nar/gkae1054

Figure Lengend Snippet: RADIP technology enables the capture of RNA–chromatin interactions mediated by a specific protein or certain types of histone marks. ( A ) Schematic of the RADIP technology. Left: Steps performed in situ on fixed nuclei after partial lysis of the nuclear membrane. Right: Steps performed in solution after disruption of the nuclear membrane by sonication. After reversal of crosslinks, the RNA–adaptor–DNA chimera is converted into a fully double-stranded DNA molecule. This molecule is then digested to a designated length using the EcoP15I enzyme. ( B ) Distribution of DNA tags from Input and H3K27me3-RADIP samples around H3K27me3 and EZH2 ChIP-seq peaks in local windows (±3 kb from the center points of the ChIP-seq peaks). ( C ) Distribution of DNA tags from Input and H3K27me3-RADIP samples around H3K9me3 ChIP-seq peaks and ATAC-seq peaks in local windows (±3 kb from the center points of the ChIP-seq peaks and ATAC-seq peaks). ( D ) Comparison of features across different technologies. ( E ) Analysis of read length and mapping results includes uniquely mapped (blue) and multiply mapped (orange) reads reported as percentages of the total read pool. For direct comparison, both DNA and RNA tags of RADICL-seq reads were artificially trimmed to 20 nucleotides. ( F ) Assessment of genomic coverage of DNA tags across different technologies in relation to sequencing depth. Note: Different technologies use various mathematical models to filter out low-frequency RNA–DNA interactions (non-specific interactions), which can influence and change the characteristics of the experimental data. Therefore, when discussing aspects such as enrichment, immunoprecipitation efficiency and comparing results across different technologies, it is crucial to use the raw data before applying background removal. This approach allows for direct comparisons and provides a clearer understanding of the experimental benefits at a fundamental level. All panels were generated by using datasets before the background removal step.

Article Snippet: In brief, 20 million crosslinked cells were resuspended in 4 ml of ice-cold lysis buffer {10 mM Tris-HCl (pH = 8.0), 10 mM NaCl, 0.2% Igepal CA-630 [octylphenoxy poly(ethyleneoxy)ethanol], 1 mM phenylmethylsulfonyl fluoride (PMSF), 1× cOmplete Protease Inhibitor Cocktail (Roche), 0.8 U/μl RNasin Plus (Promega)} and incubated on ice for 10 min. Chimeric molecules of RNA–internal bridge adaptor–DNA were generated with 2 million cells per tube; multiple tubes were processed in parallel before the sonication step and then pooled; 0.122 U/μl RNase H (New England Biolabs) was added and the reaction was incubated at 37°C for 40 min before the RNA–internal bridge adaptor ligation step following a published protocol ( ).

Techniques: In Situ, Lysis, Membrane, Disruption, Sonication, ChIP-sequencing, Comparison, Sequencing, Immunoprecipitation, Generated