DOI: 10.5061/dryad.4xgxd257p
title: Dual DNA/protein tagging of open chromatin unveils dynamics of epigenomic landscapes in leukemia

description: The architecture of chromatin specifies eukaryotic cell identity by
 controlling transcription factor access to sites of gene regulation. Here
 we describe a dual transposase/peroxidase approach, integrative DNA And
 Protein Tagging (iDAPT), which detects both DNA (iDAPT-seq) and protein
 (iDAPT-MS) associated with accessible regions of chromatin. In addition to
 direct identification of bound transcription factors, iDAPT enables the
 inference of their gene regulatory networks, protein interactors, and
 regulation of chromatin accessibility. We applied iDAPT to profile the
 epigenomic consequences of granulocytic differentiation of acute
 promyelocytic leukemia, yielding previously undescribed mechanistic
 insights with potential therapeutic implications. Our findings demonstrate
 the power of iDAPT as a discovery platform for both the dynamic epigenomic
 landscapes and their transcription factor components associated with
 biological phenomena and disease.
description: ATAC-see
 was performed similarly as previously described with slight
 modifications<sup>19</sup>. Enzyme and transposon DNA were
 mixed at a 1:1.25 enzyme:MEDS-A/B-AF647 molar ratio and incubated at room
 temperature for 1 h. Adherent cells were grown on glass coverslips (Fisher
 Scientific, 12-540A) until 80-90% confluent, washed with 1xPBS, fixed with
 1% formaldehyde (Electron Microscopy Services) in 1xPBS for 10 min, and
 washed twice with ice-cold 1xPBS. Immobilized cells were lysed by
 incubation with LB1 for 3 min followed by LB2 for 10 min at room
 temperature. Cells were then subject to tagmentation (20%
 dimethylformamide, 10 mM MgCl<sub>2</sub>, 20 mM Tris-HCl pH
 7.5, 33% 1xPBS, 0.01% digitonin, 0.1% Tween-20, and 80 pmol enzyme
 equivalent of enzyme:DNA complex in a total volume of 100 μL) for 30 min
 at 37 °C in a humidified chamber. Subsequently, cells were washed with 50
 mM EDTA and 0.01% SDS in 1xPBS three times for 15 min each at 55 °C, lysed
 for 10 min with 0.5% Triton X-100 in 1xPBS at room temperature, and
 blocked with 1% BSA and 10% goat serum in PBS-T (1xPBS and 0.1% Tween-20)
 for 1 h in a humidified chamber. Primary antibody was added to slides in
 1% BSA/PBS-T and incubated at 4 °C overnight; slides were then washed and
 subjected to secondary antibody staining for 1 h. Slides were washed with
 PBS-T three times for 15 min each, stained with DAPI (Sigma, 1 μg/mL) for
 1 min, washed with PBS for 10 min, and mounted with Fluorescence Mounting
 Medium (Dako). Confocal microscopy images were taken with an LSM 880 Axio
 Imager 2 or an LSM 880 Axio Observer at 63x magnification (Zeiss). Images
 were processed with Fiji/ImageJ
 v2.0.0.      
 Primary antibodies used were anti-RNA polymerase II CTD repeat YSPTSPS
 (phospho S2) (rabbit, Abcam ab5095, 1:500), anti-H3K27Ac (rabbit, Abcam
 ab4729, 1:500), anti-H3K9me3 (rabbit, Abcam ab8898, 1:500), anti-SC35
 (mouse, SC-35, Abcam ab11826, 1:1000). Secondary antibodies used were Goat
 anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate
 (Thermo Fisher Scientific A11008, 1:1000) and Goat anti-Mouse IgG (H+L)
 Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 conjugate (Thermo
 Fisher Scientific A11001, 1:1000).