In situ Chromatin Interaction Analysis Using Paired‐End Tag Sequencing

Abstract

Chromatin Interaction Analysis Using Paired‐End Tag Sequencing (ChIA‐PET) is an established method to map protein‐mediated chromatin interactions. A limitation, however, is that it requires a hundred million cells per experiment, which hampers its broad application in biomedical research, particularly in studies in which it is impractical to obtain a large number of cells from rare samples. To reduce the required input cell number while retaining high data quality, we developed an in situ ChIA‐PET protocol, which requires as few as 1 million cells. Here, we describe detailed step‐by‐step procedures for performing in situ ChIA‐PET from cultured cells, including both an experimental protocol for sample preparation and data generation and a computational protocol for data processing and visualization using the ChIA‐PIPE pipeline. As the protocol significantly simplifies the experimental procedure, reduces ligation noise, and decreases the required input of cells compared to previous versions of ChIA‐PET protocols, it can be applied to generate high‐resolution chromatin contact maps mediated by various protein factors for a wide range of human and mouse primary cells. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample preparation and data generation Support Protocol: Bridge linker preparation Basic Protocol 2: Data processing and visualization

ChIATAC is an efficient strategy for multi-omics mapping of 3D epigenomes from low-cell inputs

“…Our main objective was to develop a method capable of mapping chromatin interactions that reveal transcriptional regulatory programs in samples of a few thousand cells, thereby enabling studies of chromatin interactions to be applied to a broader range of biological and clinical samples. Inspired by the high efficiency and simplicity of the ATAC-seq protocol for mapping open chromatin loci 15 , we designed a strategy for mapping chromatin interactions that combines the proximity ligation of in situ Hi-C 7 and in situ ChIA-PET 21 with the Tn5-mediated in situ digestion of ATAC-seq 15 for simultaneous mapping of chromatin interactions and open chromatin loci with the goal of reducing the required input cells down to a few thousands or even hundreds, without compromising data quality. We call this method ChIA-ATAC, or in short, ChIATAC.…”

“…We call this method ChIA-ATAC, or in short, ChIATAC. In ChIATAC, cells are crosslinked, and the intact but permeabilized nuclei are subjected to in situ restriction enzyme digestion and followed by proximity ligation with a biotinylated bridge linker, as described in the in situ ChIA-PET protocol 21 . The nuclei are then processed for in situ transposase-based tagmentation, as established in the ATAC-seq protocol 15 .…”

“…In situ ChIA-PET libraries with 20 μg antibodies against RNAPII (Biolegend; 920102) or CTCF (ABclonal; A1133) were constructed using 10,000,000 FA-EGS-crosslinked cells from GM12878 cell cultures, following the in situ ChIA-PET protocol 21 . The ChIA-PET libraries were sequenced by paired-end sequencing (2 × 150 bp) on an Illumina NovaSeq 6000.…”

“…Improving efficiency and reducing the number of input cells for broader application of chromatin interaction assays has been challenging 21 . Here, we report a strategy called ChIATAC that combines in situ proximity ligation for chromatin interaction analysis (ChIA) and the efficiency of transposase-based ATAC approach for chromatin library construction.…”

Epigenetic activation of the FLT3 gene by ZNF384 fusion confers a therapeutic susceptibility in acute lymphoblastic leukemia

“…In situ ChIA-PET libraries with antibodies against RNAPII (BioLegend, #664906, 20 μg), CTCF (Abclonal, #A1133, 20 μg), and ZNF384 (Abclonal, #A15964, 20 μg) were generated using ~10 million input Article https://doi.org/10.1038/s41467-022-33143-w cells from JIH5 and TCF3-ZNF384 ALL PDX cells, following the in situ ChIA-PET protocol 51 , with the following steps: (1) cells were crosslinked by standard formaldehyde treatment, and the pellets were then snapfrozen in liquid nitrogen; (2) the crosslinked cells were lysed to release the chromatin-DNA complexes followed by fragmentation to an average size of 300 base pairs. (3) The sonicated chromatin-DNA complexes were incubated with the antibody-coated magnetic Protein G beads (Covance, #8WG16).…”