ChIP-Seq identifies DNA-associated protein binding sites genome-wide. ChIP-seq starts withchromatin immunopreciptation, including  crosslinking DNA-protein complexes, fragmenting and treating the DNA with an exonuclease to trim unbound oligonucleotides. Protein-specific antibodies immunoprecipitate the DNA-protein complex, and DNA extraction and sequencing yield high-resolution protein-binding site sequences.

While the CGI does not offer ChIP as a service due to the variability in sample types and queried proteins, we perform library preparation and Illumina sequencing on user-prepared ChIP samples. Based on a 3GB haploid genome like human, we recommend 40-50M PE reads for marks broadly distributed across the genome, such as modified histones (e.g. H3K27me3, H3K36me3, RNA Pol II, H3K9me3). Only 20M PE reads for marks confined to narrower genomic regions (e.g. transcription factors, H3K4me, H3K27ac). Reads can be scaled for larger or smaller genomes based on these recommendations. 

Note that you should use an antibody that has been successfully validated for ChIP-seq. For a new sample/antibody combination, we recommend a pilot study to estimate required sequencing depth. Depending on your statistical analysis workflow, it is recommended you perform an input and/or IgG control with each sample-antibody combination. 

CUT&RUN (Cleavage Under Targets and Release Using Nuclease) and CUT&Tag (Cleavage Under Targets and Tagmentation) are two antibody-targeted chromatin profiling methods to measure the histone modification enrichment or transcription factor binding. Both offer several advantages over traditional ChIP-seq. Both techniques require lower cell numbers/less starting material, involve less sample manipulation thereby reducing the risk of potential artifacts, higher resolution and greater specificity with less background noise, and is a more cost-effective option. 

CUT&RUN: The procedure is carried out in situ where micrococcal nuclease tethered to protein A binds to an antibody of choice and cuts immediately adjacent DNA, releasing DNA-bound to the antibody target. CUT&RUN provides transcription factor or histone modification profiles while avoiding crosslinking and solubilization issues typical in ChIP-seq. Extremely low backgrounds make profiling possible with less sequencing depth than is required for ChIP-seq and permits profiling using low cell numbers without losing quality. This method is recommended for profiling transcription factor binding.

CUT&Tag: This method generates sequence-ready libraries without the need for end polishing and adapter ligation. It uses a proteinA-Tn5 fusion to tether Tn5 transposase near the site of an antibody to a chromatin protein of interest. A secondary antibody is used to increase the efficiency of tethering the pA-Tn5 to the target primary antibody. The pA-Tn5 complex is pre-loaded with sequencing adapters that insert into adjacent DNA upon activation with magnesium. CUT&Tag has a very low background and can be performed in a single tube in a day, though primary antibodies are typically incubated overnight. This method is recommended for profiling histone marks and RNA polII. 

CUT&Tag-direct: A streamlined CUT&Tag protocol was introduced by the Henikoff Lab that suppresses DNA accessibility artifacts to ensure high-fidelity mapping of the antibody-targeted protein and improves the signal-to-noise ratio over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube, from cells to amplified libraries, providing low-cost genome-wide chromatin maps. By simplifying library preparation, CUT&Tag-direct requires less than a day of benchwork, from live cells to sequencing-ready barcoded libraries. 

While the CGI does not offer CUT&RUN/Tag as a service due to the variability in sample types and queried proteins, we perform library preparation and Illumina sequencing on user-prepared samples. For a new sample/antibody combination, we recommend a pilot study to estimate required sequencing depth. Depending on your statistical analysis workflow, it is recommended you perform an input and/or IgG control with each sample-antibody combination. We recommend 20-30M PE reads (based on a 3GB genome), noting that less coverage may be adequate. 

DiMeLo-seq (Directed Methylation with Long-read sequencing) is a single-molecule technique for mapping protein-DNA interactions across native chromatin without the need for crosslinking, sonication, or immunoprecipitation. The method uses an antibody targeted to a protein of interest, tethered to a methyltransferase enzyme, to deposit exogenous adenine methylation (m6A) marks on DNA in close proximity to the protein’s binding sites. Because these m6A marks are not naturally abundant in most eukaryotic genomes, they serve as a distinct, orthogonal readout of protein binding that can be directly detected using long-read sequencing platforms such as Oxford Nanopore, without additional chemical conversion.

This approach preserves long-range genomic context, enabling simultaneous mapping of protein binding, nucleosome positioning, and endogenous CpG methylation on the same DNA molecule. Moreover, the technique is particularly powerful for studying multi-protein co-occupancy, allele-specific binding, and chromatin states in repetitive (e.g. centromeres) or structurally complex genomic regions that are difficult to resolve with traditional short-read methods like ChIP-seq.

While the CGI does not offer DiMeLo-seq as a service due to the variability in sample types and queried proteins, we can provide protocols and advice on experimental design. We can perform library preparation and ONT sequencing on the PromethION on user-prepared samples.

ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a technique to map genome-wide chromatin accessibility. Open, active regions of chromatin across the genome represent regions that are involved in gene expression, such as transcription factor binding sites and cis-regulatory elements. In this method, the genome is fragmented with a hyperactive transposase Tn5, which is pre-loaded with sequencing adaptors. Simultaneous fragmentation of chromatin into short fragments (<500bp) and integration of sequencing adaptors into regions of open chromatin result in a DNA pool that is amplified/library prepped for sequencing on an Illumina instrument. 

The CGI performs QC, library preparation and Illumina sequencing on user-prepared samples. We recommend 50-60M PE reads (based on a 3GB genome).

Fiber-seq is a method developed by the Stergachis lab uses PacBio or ONT sequencing platforms to assess chromatin accessibility using long-read sequencing. Nuclei are treated with Hia5 N6– methyladenine methyltransferase (6mA MTase) (available from CANTANA) which labels accessible adenines with 6mA, effectively “stenciling” chromatin accessibility directly onto the DNA. gDNA is then purified and prepped for sequencing on either the ONT or PacBio platform, affording detection of chromatin accessibility, DNA methylation and genetic information (i.e. variants) on each DNA molecule sequenced. This multi-omic information is mapped to a genome assembly, providing single nucleotide and single molecule resolution across the genome, including difficult to traverse regions, such as repetitive regions.

 While the CGI does not offer DFiber-seq as a service due to the variability in sample types and quality, we can provide protocols and advice on experimental design. We can perform library preparation and sequencing on the ONT PromethION or PacBio Revio on user-prepared samples.