CUT&RUN: Targetedin situgenome-wide profiling with high efficiency for low cell numbers

Abstract

SUMMARYCleavage Under Targets and Release Using Nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widelyused Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and can be used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&RUN requires less than a day at the lab bench.

Non-B-Form DNA Is Enriched at Centromeres

“…CUT&RUN was performed as described ( Skene and Henikoff 2017a , b ). In brief, K562 or Cos-7 cells were gently washed twice in room temperature Wash buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and a Roche complete EDTA-free tablet (Sigma-Aldrich) per 50 ml], with 3 min centrifugation at 600×g, mixed with activated Concanavalin A-coated magnetic beads (Bangs Laboratories) and rotated 5–10 min.…”

Unexpected conformational variations of the human centromeric chromatin complex

“…We recently introduced CUT&RUN, an efficient targeted nuclease method that is unrelated to ChIP in that it causes precise cleavage and release of intact antibody targeted particles without solubilizing the rest of the genome (Skene and Henikoff 2017b). In our most recent CUT&RUN protocol (Skene and Henikoff 2017a), antibodies are added to permeabilized cells bound to magnetic beads followed by addition of a protein fusion between MNase and protein A (pA-MN), which binds to the antibody. MNase is activated by calcium and then stopped by chelation with EDTA and EGTA in the presence of 175 mM NaCl.…”

“…Experiments shown in Figure 4 and Supplemental Table 1 used permeabilized cells rather than nuclei (Skene and Henikoff 2017a). Paired-end 250-bp × 250-bp or 25-bp × 25-bp sequencing was performed by the Fred Hutch Shared Genomics Resource.…”

Hhex Directly Represses BIM-Dependent Apoptosis to Promote NK Cell Development and Maintenance

“…Remarkably, the proportion and total numbers of NK cells in all hematopoietic tissues of Hhex D/D Bim D/D mice were comparable with Cre control mice (Figure 4A), confirming BIM-mediated apoptosis as the key driver of NK cell lymphopenia in Hhex -D /D mice. In order to investigate if HHEX directly inhibits BIM expression by binding to the Bcl2l11 gene, we performed cleavage under targets and release using nuclease (CUT&RUN [CnR]) sequencing, a more sensitive technique that is based on the main principles of chromatin immunoprecipitation (ChIP) sequencing (Skene and Henikoff, 2017). GREAT analysis of peaks called by MACS2 revealed 1,554 genes in the proximity (within 0.1 kB) of HHEX binding sites.…”