4DN Protocols

Collection of genomic technologies currently in use or being developed in the 4DN network.

4DN Steering Committee approved protocols
Protocol Date of approval Description
iMARGI August 18, 2020 in situ Mapping of RNA-chromatin interactions (iMARGI) protocol for mapping of chromatin-associated RNAs.
PLAC-seq/HiChIP/in situ ChIA-PET August 18, 2020 PLAC-seq/HiChIP/in situ ChIA-PET protocols to detect and quantify chromatin contacts anchored at genomic regions associated with specific DNA binding proteins or histone modifications.
Single-cell Hi-C Protocol April 17, 2018 Single-cell Hi-C Protocol and Quality Control Standards
DNase Hi-C protocol January 16, 2018 Mapping 3D genome architecture through in situ DNase Hi-C
DamID seq protocol December 12, 2017 The Official 4DN Standard DAM-ID seq Protocol from the van Steensel Lab.
ChIA-PET data analysis October 17, 2017 ChIA-PET data processing pipeline and standards, 4D Nucleome Consortium
ChIA-PET experimental protocol October 17, 2017 ChIA-PET Protocol and Standards, 4D Nucleome Consortium
E/L Repli-seq June 20, 2017 E/L Repli-seq: Protocol and Quality Control Standards, 4D Nucleome Consortium
E/L Repli-seq June 20, 2017 4D Nucleome Consortium, Overall Standards and Guidelines for E/L Repli-seq Experiments
Hi-C February 21, 2017 Standards and Guidelines for Hi-C experiments
In situ Hi-C February 21, 2017 Protocol and Quality Control Standards for in situ Hi-C experiments


Other relevant protocols
Technology (Protocol) Reference paper Description
3C-seq Capturing Chromosome Conformation (Dekker et al, Science, 2002) Chromosome conformation capture techniques are used to analyze the organization of chromatin in a cell by quantifying the interactions between genomic loci that are nearby in 3-D space. 3C quantifies interactions between a single pair of genomic loci (one-vs-one).
4C-seq 4C technology: protocols and data analysis (Van de Werken et al, Methods Enzymol, 2012) Chromosome conformation capture-on-chip (4C) captures interactions between one locus and all other genomic loci (one-vs-all).
5C-seq Mapping networks of physical interactions between genomic elements using 5C technology (Josée Dostie and Job Dekker, Nature Protocols, 2007) Chromosome conformation capture carbon copy (5C) detects interactions between all restriction fragments within a given region, with this region's size typically no greater than a megabase (many-vs-many).
Capture Hi-C Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C (Mifsud et al, Nature Genetics, 2015) Capture Hi-C (CHi-C) is is an adapted technology that selects and enriches few hundred promoters for genome-wide, long-range contacts of both active and inactive promoters.
ChIA-PET ChIP-based methods for the identification of long-range chromatin interactions (Fullwood and Ruan, Journal of Cellular Biochemistry, 2009) Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) is a technique that incorporates chromatin immunoprecipitation (ChIP)-based enrichment, chromatin proximity ligation, Paired-End Tags, and High-throughput sequencing to determine de novo long-range chromatin interactions genome-wide.
COLA Deletion of DXZ4 on the human inactive X chromosome alters higher-order genome architecture (Darrow et al., PNAS, 2016) COLA (Concatamer Ligation Assay) is a modified in situ Hi-C protocol that uses CviJI restriction enzyme to digests chromatin into much finer fragments the original Hi-C method, in order to increase the proportion of reads containing three or more nearby fragments.
CUT&RUN An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites (Skene and Henikoff, eLIFE, 2017) CUT&RUN is an efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is an antibody-targeted chromatin profiling method in which micrococcal nuclease tethered to protein A binds to an antibody of choice and cuts immediately adjacent DNA, releasing DNA bound to the antibody target. The procedure is carried out in situ and produces precise transcription factor or histone modification profiles while avoiding crosslinking and solubilization issues. Extremely low backgrounds make profiling possible with typically one tenth of the sequencing depth required for ChIP, and permit profiling using low cell numbers without loss of quality. CUT&RUN can also be used to map long-range genomic contacts.
DamID Identification of in vivo DNA targets of chromatin proteins using tethered Dam methyltransferase (Bas van Steensel and Steven Henikoff, Nature, 2000) DamID enables mapping genome-wide occupancy of interaction sites in vivo, based on the expression of a fusion protein consisting of a protein of interest and DNA adenine methyltransferase (Dam). This leads to methylation of adenines near sites where the protein of interest interacts with the DNA. These methylated sequences are subsequently amplified by a methylation-specific PCR protocol and identified by hybridization to microarrays.
Dnase Hi-C Mapping 3D genome architecture through in situ DNase Hi-C (Ramani et al., Nature Protocols, 2016) DNAse Hi-C complements high resolution Hi-C approach with restriction enzymes.
Hi-C Comprehensive Mapping of Long-Range Interactions Reveals Folding Principles of the Human Genome (Lieberman-Aiden et al. Science, 2009) Hi-C uses high-throughput sequencing to find the nucleotide sequence of fragments ( all-vs-all).
Hi-C^2 Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes (Sanborn et al., PNAS, 2015) Hybrid Capture Hi-C (Hi-C^2) is a technology that combines targeted genomic capture and existing situ Hi-C libraries to observe conformation changes in selected genomic regions.
iMARGI Mapping RNA–chromatin interactions by sequencing with iMARGI (Wu, Yan, et al., Nat Protocols, 2019) iMARGI (in situ mapping of RNA-Genome Interactome) is a technique that globally maps native RNA-genome interactions from unperturbed cells. It is an improved version of traditional MARGI published in 2017.
In situ Hi-C A 3D Map of the Human Genome at Kilobase Resolution Reveals Principles of Chromatin Looping (Rao et al., Cell, 2014) In situ Hi-C method is used to evaluate all DNA-DNA proximity ligation in intact nuclei.
Micro-C Micro-C XL: assaying chromosome conformation from the nucleosome to the entire genome (Hsieh et al., Nature Methods, 2016) Micro-C enables mono nucleosome-resolution analysis of chromosome folding fragmentation using micrococcal nuclease (MNase). Micro-C XL is implemented by adding long x-linkers to the fragments.
NLA Interaction between transcription regulatory regions of prolactin chromatin (Cullen et al., Science, 1993) Nuclear Ligation Assay (NLA) is an historical method developped in 1993 to determine circularization frequencies of DNA in solution. It inspired 3C method.
PLAC-seq Proximity Ligation Assisted ChIP-seq (PLAC-seq), also known as HiChIP, combines in situ proximity ligation with chromatin immunoprecipitation (ChIP) to map chromatin interactions centered on genomic regions bound by the transcription factor or histone with the specific modifications with much reduced cost compared to Hi-C.
Repli-Seq Repli-seq: genome-wide analysis of replication timing by next-generation sequencing (Marchal et al., bioRxiv, 2017) Repli-Seq is a genome-scale approach to map temporally ordered replicating DNA using massively parallel sequencing.
Single-Cell DamID Single-Cell Dynamics of Genome-Nuclear Lamina Interactions (Kind et al., Cell, 2013) Single Cell DamID enables visualization of in vivo with adenine-6-methylation of intact cells by couopling DamID technique and engineered DpnI digestion enzyme.
Sci-Hi-C Sci-Hi-C: A single-cell Hi-C method for mapping 3D genome organization in large number of single cells (Ramani et al., Methods, 2020) Single-cell combinatorial indexed Hi-C (Sci-Hi-C) is a high throughput method that enables mapping chromatin interactomes in large number of single cells.
Single-cell Hi-C Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell (Nagano et al., Nature Protocols, 2015) Single Cell Hi-C is an adaptation of Hi-C to single-cell analysis, by including in-nucleus ligation.
Split-pool Barcoding Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets (Macosko et al., Cell, 2015) Split-pool barcoding or Drop-seq is a strategy for profiling thousands of individual cells by separating them into nanoliter-sized aqueous droplets and associating a different barcode with each cell's RNAs and sequencing them all together.
TRIP Using TRIP for genome-wide position effect analysis in cultured cells (Akhtar et al., Nature Protocols, 2014) This is protocol for analyzing thousands of reporters integrated in parallel (TRIP) at a genome-wide level. TRIP is based on tagging each reporter with a unique barcode, which is used for independent reporter expression analysis and integration site mapping?