Universal NicE-seq for high-resolution accessible chromatin profiling for formaldehyde-fixed and FFPE tissues

Abstract

Accessible chromatin plays a central role in gene expression and chromatin architecture. Current accessible chromatin approaches depend on limited digestion/cutting and pasting adaptors at the accessible DNA, thus requiring additional materials and time for optimization. Universal NicE-seq (UniNicE-seq) is an improved accessible chromatin profiling method that negates the optimization step and is suited to a variety of mammalian cells and tissues. Addition of 5-methyldeoxycytidine triphosphate during accessible chromatin labeling and an on-bead library making step substantially improved the signal to noise ratio while protecting the accessible regions from repeated nicking in cell lines, mouse T cells, mouse kidney, and human frozen tissue sections. We also demonstrate one tube UniNicE-seq for the FFPE tissue section for direct NGS library preparation without sonication and DNA purification steps. These refinements allowed reliable mapping of accessible chromatin for high-resolution genomic feature studies.

One-pot universal NicE-seq: all enzymatic downstream processing of 4% formaldehyde crosslinked cells for chromatin accessibility genomics

“…To capture biotin-labeled DNA, 30 µl of magnetic streptavidin beads (NEB, S1420S) were added and incubated at 4 °C for 2 h in the presence of 2 M NaCl. Then the library was prepared as mentioned in Chin et al [ 16 ] with some modifications. Briefly, after the incubation of biotin-labeled DNA with magnetic streptavidin beads, the beads were washed twice with high salt buffer (10 mM Tris–HCl pH = 8, 2 M NaCl, 1 mM EDTA and 0.05% TritonX-100) and once with 1× TE buffer.…”

“…Then the beads were resuspended in 50 µl of 0.1× low TE buffer. Then end-repair/dA tailing was done in a thermo cycler as mentioned in Chin et al [ 16 ]. Then the beads were washed twice with 200 µl high salt buffer and once with 1× TE.…”

“…Then the beads were resuspended in 60 µl of 0.1× low TE buffer. Then the adapter ligation, PCR amplification and library clean-up was done as mentioned in Chin et al [ 16 ]. Sequencing was done on Illumina Nextseq 550 system.…”

“…Read processing and data analysis was done as mentioned in Chin et al [ 16 ] and Estève et al [ 17 ]. Briefly, adapter and low-quality sequences from the raw FASTQ files were trimmed using Trim Galore ( http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/ ).…”

“…The addition of 5mdCTP prevented further nicking and degradation of accessible chromatin DNA. Universal NicE-seq typically uses formaldehyde to cross-link chromatin, and an undeniably powerful approach for determining chromatin accessibility in culture cells, frozen tissue sections, and FFPE sections with relatively intact DNA [ 16 , 17 ].…”