ChIL-seq, Chromatin Integration Labelling followed by sequencing is an epigenomic profiling method which enables the amplification of genomic sequences closely associated with the target molecules before cell lysis. ChIL-seq is particularly useful for epigenomic profiling of low input samples (100 to 100K cells, thin tissue sections) and it is suitable for single cell studies. It allows to study a wide range of histone modifications and transcription factors with results comparable to ChIP-seq or CUT&Tag. Moreover, this technology is using a fluorescent probe allowing spatial localization under a microscope and the control of the immunostaining reaction.
Diagenode’s ChIL-seq protocol for cells has been adapted from the classic ChIL-seq protocol developed by Professors Ohkawa and Kimura in Japan, first published in Nature Cell Biology in 2019 then in Nature Protocols in 2020 (Ref. 1 and 2). The key component of the protocol, a ChIL probe (which comprises the secondary antibody (anti-mouse or anti-rabbit IgG) conjugated with double- stranded DNA (ChIL DNA) containing a T7 promoter and a primer sequence for the sequencing library preparation, including a mosaic end for Tn5 transposase binding) is finally available from Diagenode: ChIL probe (goat, anti-rabbit) and ChIL probe (rat, anti-mouse). Other Diagenode’s products have been validated with the ChIL-seq protocol, including: Tagmentase (unloaded), Tagmentase Dilution Buffer, H3K4me4 and H3K27me3 positive control antibodies, rabbit and mouse IgG negative control antibodies, Primer indexes for tagmented libraries.
- How does it works? - Read more
ChIL protocol involves growing cells on a 96-well plate and fixing, permeabilizing and staining with the primary antibody, as in the standard immunostaining procedure, before incubation with the ChIL probe and in situ localization by immunofluorescence. The ChIL probe comprises the secondary antibody (anti-mouse or anti-rabbit IgG) conjugated with double- stranded DNA (ChIL DNA) containing a T7 promoter and a primer sequence for the sequencing library preparation, including a mosaic end for Tn5 transposase binding. ChIL DNA is integrated into a proximal genomic region of the primary antibody’s target through Tn5 transposase and the integrated ChIL DNA is then used for linear amplification of the genomic sequences following the sequencing primer, by in situ transcription with T7 RNA polymerase. The transcribed RNA molecules are then used to prepare libraries for deep sequencing and the sequenced reads are mapped to the appropriate reference genome for further analysis.
ChIL-seq features:
- Sample requirement: 100 – 100K adherent cells
- Visualisation of the spatial localization
- Chromatin preparation-free method
- Robust library preparation – by applying an antibody-targeted controlled in situ transposition by Tn5 to link the adaptors
- Decreased PCR duplicates & increased unique reads due to the use of a linear amplification by the T7 RNA polymerase
- Allows massively parallel DNA-sequencing because of the 96-wells format
Watch our webinar: "ChIL technology for lower input epigenomics with high genome coverage":
References
1. Harada, A., Maehara, K., Handa, T. et al. A chromatin integration labelling method enables epigenomic profiling with lower input. Nat Cell Biol 21, 287–296 (2019). https://doi.org/10.1038/s41556-018-0248-3
2. Handa, T., Harada, A., Maehara, K. et al. Chromatin integration labeling for mapping DNA-binding proteins and modifications with low input. Nat Protoc 15, 3334–3360 (2020). https://doi.org/10.1038/s41596-020-0375-8
3. Maehara K, Tomimatsu K, Harada A, Tanaka K, Sato S, Fukuoka M, Okada S, Handa T, Kurumizaka H, Saitoh N, Kimura H, Ohkawa Y. Modeling population size independent tissue epigenomes by ChIL-seq with single thin sections. Mol Syst Biol. 2021 Nov;17(11). https://doi.org/10.15252/msb.202110323
