GSM7947622: KO_T0_H2AK118ac_3; Homo sapiens; OTHER
E. coli (C3013, New England Biolabs) was transformed with plasmid pET15bpATnp (#121137, Addgene), the pA‐Tn5ase protein was isolated. The pA‐Tn5ase was mixed with Tn5ME‐A+B load adaptor mix in complex formation buffer (CB) to form the pA‐Tn5 transposome (pATn5ome). For every reaction, the pA‐Tn5ome‐antibody (pATn5ome‐ab) complex was formed by mixing 1 μL pA‐Tn5ome with 0.8 μL CB and 0.8μL antibody solution. The antibodies used in this study were: Rabbit polyclonal anti-acetyl-Histone H3 (Lys27) (ab4729, Abcam), Rabbit polyclonal anti-acetyl-Histone H2A (Lys118) (PTM-173, PTM BIO), and rabbit IgG (cat #pp64 Millipore, Burlington, MA, USA). Approximately 50,000 nuclei of cell were used for pA‐Tn5ome‐ab complex binding and tagmentation. For normalization of sequence reads between biological replicates, approximately 3000 permeabilized nuclei of yeast Saccharomyces cerevisiae were prepared, incubated with pATn5ome‐ ab complex targeting yeast H2B, and were spiked into each mix of cells and pATn5ome‐ab complex. Tagmented DNA was purified with a MinElute kit (#28004, Qiagen, Hilden, Germany) and eluted with 20 μL elution buffer (EB). Sequencing libraries were generated under real‐time conditions with a LightCycler 480 (Roche, Basel, Switzerland) in a reaction mix consisted of tagmented DNA eluate, NEBNext High Fidelity 2X Mix (#M0541, New England Biolabs), 100X SYBRGreen, primer Tn5McP1n, and Custom Nextera PCR Barcode. Libraries were purified with AMPure XP beads (#A63880, Beckman, Brea, CA, USA) with a bead:DNA ratio of 1.4:1. Quantity and fragment size of the libraries were determined with a Qubit dsDNA HS assay kit (#Q32854, ThermoFisher and a TapeStation 4150 with D1000 High Sensitivity Assay (#5067‐ 5585, Agilent, Santa Clara, CA, USA), respectively.