Journal Article

Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input

Nakao, Masaru, Goto, Naoki, Ohkawa, Yasuyuki, Maehara, Kazumitsu, Kimura, Hiroshi, Handa, Tetsuya, Harada, Akihito, Kurumizaka, Hitoshi, Sato, Shoko

2020

Overview

Cell identity is determined by the selective activation or silencing of specific genes via transcription factor binding and epigenetic modifications on the genome. Chromatin immunoprecipitation (ChIP) has been the standard technique for mapping the sites of transcription factor binding and histone modification. Recently, alternative methods to ChIP have been developed for addressing the increasing demands for low-input epigenomic profiling. Chromatin integration labeling (ChIL) followed by sequencing (ChIL-seq) has been demonstrated to be particularly useful for epigenomic profiling of low-input samples or even single cells because the technique amplifies the target genomic sequence before cell lysis. After labeling the target protein or modification in situ with an oligonucleotide-conjugated antibody (ChIL probe), the nearby genome sequence is amplified by Tn5 transposase-mediated transposition followed by T7 RNA polymerase-mediated transcription. ChIL-seq enables the detection of the antibody target localization under a fluorescence microscope and at the genomic level. Here we describe the detailed protocol of ChIL-seq with assessment methods for the key steps, including ChIL probe reaction, transposition, in situ transcription and sequencing library preparation. The protocol usually takes 3 d to prepare the sequencing library, including overnight incubations for the ChIL probe reaction and in situ transcription. The ChIL probe can be separately prepared and stored for several months, and its preparation and evaluation protocols are also documented in detail. An optional analysis for multiple targets (multitarget ChIL-seq) is also described. We anticipate that the protocol presented here will make the ChIL technique more widely accessible for analyzing precious samples and facilitate further applications. The authors describe detailed procedures for an epigenomic profiling method suitable for low-input samples that is based on in situ labeling with an oligonucleotide-conjugated antibody.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 631/1647/2210/2211 / 631/1647/245/2225 / 631/61/212/177 / Affinity labeling / Analytical Chemistry / Animals / Antibodies / Binding / Biological Techniques / Biomedical and Life Sciences / Cell Line / Cell Line, Tumor / Chromatin / Chromatin - metabolism / Chromatin Immunoprecipitation - methods / Chromatin Immunoprecipitation Sequencing - methods / Chromosome Mapping - methods / Computational Biology/Bioinformatics / Deoxyribonucleic acid / DNA / DNA binding proteins / DNA-binding protein / DNA-Binding Proteins - analysis / DNA-directed RNA polymerase / Epigenesis, Genetic - genetics / Epigenetics / Epigenomics - methods / Fluorescence / Gene Library / Gene mapping / Gene silencing / Genetic screening / Genome / Genomes / Genomics / High-Throughput Nucleotide Sequencing - methods / Histones / Histones - metabolism / Humans / Immunoprecipitation / Integration / Labeling / Libraries / Life Sciences / Localization / Lysis / Methods / Mice / Microarrays / Nucleotide sequence / Oligonucleotides / Organic Chemistry / Peptide mapping / Properties / Protein Processing, Post-Translational - genetics / Proteins / Protocol / RNA polymerase / Sequence Analysis, DNA - methods / Target detection / Transcription factors / Transcription Factors - metabolism / Transposase / Transposases - metabolism / Transposition