TY - JOUR
T1 - Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input
AU - Handa, Tetsuya
AU - Harada, Akihito
AU - Maehara, Kazumitsu
AU - Sato, Shoko
AU - Nakao, Masaru
AU - Goto, Naoki
AU - Kurumizaka, Hitoshi
AU - Ohkawa, Yasuyuki
AU - Kimura, Hiroshi
N1 - Funding Information:
We are grateful to the staff of Ohkawa Lab. The computation was carried out using the computer resource offered under the category of Intensively Promoted Projects by Research Institute for Information Technology, Kyushu University. This work was in part supported by MEXT/JSPS KAKENHI (JP17K17719 to T.H.; JP18K19432, JP19H03211, JP19H05425 and JP20H05368 to A.H.; JP18H04904, JP19H04970, JP19H03158 and JP20H05393 to K.M.; JP18H05534 to H.Ku.; JP18H04802, JP18H05527, JP19H05244, JP17H03608, JP20H00456, JP20H04846 and JP20K21398 to Y.O.; and JP18H05527 and JP17H01417 to H.Ki), JST CREST (JPMJCR16G1 to Y.O., H.Ku. and H.Ki.), JST PRESTO JPMJPR19K7 to A.H, AMED JP20ek0109489h0001 to Y.O. and AMED BINDS (JP19am0101076 to H.Ku. and JP19am0101105 to H.Ki.).
Publisher Copyright:
© 2020, The Author(s), under exclusive licence to Springer Nature Limited.
PY - 2020/10/1
Y1 - 2020/10/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41596-020-0375-8
DO - 10.1038/s41596-020-0375-8
M3 - Article
C2 - 32807906
AN - SCOPUS:85089514041
VL - 15
SP - 3334
EP - 3360
JO - Nature Protocols
JF - Nature Protocols
SN - 1754-2189
IS - 10
ER -