Low‐Input MNase Accessibility of Chromatin (Low‐Input MACC)

Abstract

An understanding of the dynamic structural properties of chromatin requires techniques that allow the profiling of regions of both open and closed chromatin as well as the assessment of nucleosome occupancy. The recently developed MNase accessibility (MACC) technique allows for the simultaneous measurement of chromatin opening and compaction, as well as nucleosome occupancy, on a genome‐wide scale in a single assay. This article presents a low‐input MACC procedure that considerably extends the utility of the original MACC assay. Low‐input MACC generates high‐quality data using very low cell numbers (as few as 50 cells per titration point), making it ideal for samples obtained after fluorescence‐activated cell sorting or dissection, or in clinical settings. Moreover, low‐input MACC has significantly improved several steps of the initial method, offering a more rapid and robust methodology. © 2019 by John Wiley & Sons, Inc.

Alterations in chromatin at antigen receptor loci define lineage progression during B lymphopoiesis

“…Because of the low-abundance cell populations during B cell development, we recently developed a low-input version of the MACC assay (14) that allowed us to use ex vivo primary cells from wild-type C57BL/6 mice (Materials and Methods). The great advantage of this analysis is the possibility of interrogating chromatin accessibility changes in cell populations directly after sorting.…”

“…Low-Input MACC Assay and Library Preparation. Low-input MACC assay was performed as previously described (14). Briefly, cells were collected after cell sorting, and nuclei were extracted using 1 vol of 2× nuclei extraction buffer (0.6 M sucrose, 4 mM MgOAc 2 , 2% Triton X-100, 20 mM Hepes [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], pH 7.8, 2 mM CaCl 2 , 8 mM MgCl 2 ).…”

“…Here, we set out to identify changes in these properties at AgR loci during the development of B lymphocytes. We used a recently developed low-input version (14) of the MACC (micrococcal nuclease [MNase]-based chromatin accessibility) assay (15) that allowed for the simultaneous measurement of both chromatin opening and compaction as well as nucleosome occupancy. Measurement of all of these metrics in a single assay is a unique feature of MACC that fits the design of the current study.…”

“…Our assay has been successfully applied in both mammalian and Drosophila analyses (15,19,20). Our recent improved assay (14) was designed to profile low-abundance and rare cell populations, allowing for a profile of chromatin structure dynamics at AgR loci during B lymphopoiesis.…”