High-resolution analysis of chromatin structure by Mnase-SSP. High-resolution analysis of chromatin structure by Mnase-SSP

Proof-of-concept for Mnase-SSP: a variant of Mnase-seq. Mnase-SSP dramatically increases the representation of short fragments of nucleolytically-digested DNA, enabling simultaneous analysis of transcription factor binding and nucleosome occupancy using the same assay. We used MNase-SSP to demarcate chromatin architecture at murine promoters and at transcription factor binding sites in murine embryonic stem cells. Are results reveal heterogeneity in the binding mode of C2H2 zinc fingers like Ctcf and Rest, demonstrating that Mnase-SSP, and SSP in general, as a flexible platform for profiling nucleolytically digested DNA for MNase-seq, MNase-ChIP, or CUT&RUN with reduced bias. Overall design: Nuclei are purified, treated with varying amounts of micrococcal nuclease, and then DNA fragments are purified. DNA is melted, ligated to a 3' single-strand adapter, and then subjected to second-strand synthesis, adaptor ligation, and PCR. Please note that these libraries are non-traditional in the sense that the library prep is very different from standard Illumina protocols, and so the raw fastqs have been subjected to non-standard adaptor trimming + read filtration (in addition to read deduplication), generating BAM files as only processed data.