Nanopore assay

Affinity-purified TAP-tagged chromatin eluted from beads was concentrated using amicon ultra 0.5 ml (UFC503024; EMD Millipore) and dialyzed against TE buffer. Chromatin was then driven through solid-state nanopores (Soni and Dekker, 2012). To fabricate nanopores, silicon wafers with triple coating of 20 nm silicon nitride, 100 nm silicon oxide, and 500 nm silicon nitride were processed with standard photolithography and e-beam lithography to fabricate freestanding silicon nitride membranes (with 20 µm square shape and 20 nm thickness; Storm et al., 2003; Wu et al., 2009; van den Hout et al., 2010). A focused transmission EM beam was used to drill 20-nm-wide pores in these membranes. We mounted these chips onto a fluid cell with fluid chambers with separate access to the cis and trans sides of the nanopore membrane. The chambers were filled with buffer (1 M KCl and 10 mM Tris-EDTA, pH 8), and a 100-mV potential was applied across the nanopore membrane using Ag/AgCl electrodes. Nanopore current was measured using Axopatch 200B (Molecular Devices) set at full bandwidth (100 KHz). To detect the sample at single-molecule resolution, ∼1 pM of the nucleosome sample was introduced in the flow chamber with negative bias. Voltage-dependent translocation of individual nucleosomes through the nanopores was recorded as temporary drops in the nanopore conductance. This drop in conductance is directly proportional to the molecular volume of the nucleosome complex (Soni and Dekker, 2012). Data were recorded using a DAQ card (NI-PCI 6251; National Instruments) at acquisition rate of 200 kHz. For analysis, data were filtered at 35 kHz. All data acquisition and event detection in current traces was done by custom-written software in LabVIEW (National Instruments) and MATLAB (Mathworks Inc.), respectively (Storm et al., 2005; Soni and Dekker, 2012).