Epigenetics application guide Learn more at www.abcam.com/epigenetics 2 Contents Introduction . 6 - Why is epigenetics important? . 6 - How to study epigenetics . 6 Chromatin accessibility and architecture . 8 - Methods to study DNA accessibility and nucleosome positioning . 9 - DNase-seq . 9 - MNase-seq . .. 9 - ATAC-seq . 10 - Chromosome conformation techniques . 11 - Chromatin conformation capture (3C) . 11 - Circularized chromosome conformation capture (4C) . 12 - Carbon copy chromosome conformation capture (5C) . 12 - Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) . 12 - ChIP-loop . 13 - Hi-C . 13 - Capture-C . 13 - References . 14 Histone modifications . 15 - Histone modifications in detail . 16 - Acetylation . 16 - Methylation . 16 - Phosphorylation . 17 - Ubiquitylation . 17 - Studying histone modifications by ChIP . 18 - Histone modifying enzymes: writers and erasers . 19 - Characterizing histone methylation pathways . 20 - Characterizing demethylase activity . 20 - Characterizing histone acetylation pathways . .. 20 - Characterizing histone deacetylase activity . .. 20 - Inhibiting writers and erasers . 21 - Histone modification readers/translators . 21 - Effector domains recognize specific histone modifications . 22 - Multivalency enables histone code complexity . 22 - References . 23 Studying epigenetics with ChIP . 24 - Applications of ChIP . 24 - Combined analysis . 25 - ChIP expands the scope and precision of epigenetic research . 25 - Protocol overview: how ChIP works . 25 - Sample Preparation: X-ChIP vs N-ChIP . 27 - Chromatin fragmentation differences . 28 Learn more at www.abcam.com/epigenetics 3 - Antibody selection . 29 - Controls . 29 - Sample controls . 29 - Antibody controls . 30 - Quantitative PCR controls . 30 - Protocol optimization . 30 - ChIP with low cell numbers . 32 - ChIP from tissue . 33 - Troubleshooting . 33 - ChIP readout . 35 - qPCR . 35 - ChIP-on-Chip . 35 - ChIP-seq . 35 - Data analysis . 36 - References . 37 Chromatin profiling using CUT&RUN and CUT&Tag . 38 - Applications of CUT&RUN and CUT&Tag . 39 - Chromatin Modifications . 39 - Transcription factors and chromatin-associated complexes . 39 - Comparison of CUT&RUN and CUT&Tag with ChIP-seq . 40 - CUT&RUN protocol overview . 41 - 1 . Nuclei extraction and binding to beads . 42 - 2 . Permeabilization and antibody binding . 42 - 3 . MNase binding and cleavage of target sequences . 42 - 4 . DNA recovery . 42 - 5 . Library preparation and sequencing . 42 - CUT&Tag protocol overview . .. 44 - 1 . Nuclei extraction and binding to beads . 45 - 2 . Permeabilization and antibody binding . 45 - 3 . Tn5 binding and tagmentation . .. 45 - 4 . DNA recovery . 45 - 5 . Library preparation using PCR amplification and sequencing . 45 - Sample preparation . 46 - Antibody selection . 46 - Controls . 46 - Optimization . 47 - Permeabilizations . 47 - Antibody concentration . 47 - Duration.