![]() 1b), where permeabilized cells are first mixed with an antibody, and then immobilized on Concanavalin A-coated paramagnetic beads, allowing magnetic handling of the cells in all successive wash and reagent incubation steps. The entire protocol manipulates all steps in a single reaction tube (Fig. Finally, fragment libraries were enriched from purified DNA and pooled for multiplex paired-end sequencing on an Illumina HiSeq flow-cell. We then activated the transposome by addition of Mg ++, integrating adapters spanning sites of H3K27me3-containing nucleosomes. The transposome has inherent affinity for exposed DNA 16, 17, and so we washed cells under stringent conditions to remove un-tethered pA-Tn5. We incubated cells with a secondary anti-rabbit antibody to increase the local concentration of antibody bound on chromatin sites, then incubated cells with an excess of pA-Tn5 fusion protein pre-loaded with sequencing adapters to tether the enzyme at antibody-bound sites in the nucleus. ![]() 1a), we incubated intact permeabilized human K562 cells with an antibody to lysine-27-trimethylation of the histone H3 tail (H3K27me3), an abundant histone modification that marks silenced chromatin regions. To implement chromatin profiling by tagmentation (Fig. This easy, low-cost method will empower epigenetic studies in diverse areas of biological research.Įfficient profiling of nucleosomes and RNAPII with CUT&Tag We show that a variety of chromatin components can be profiled with exceptionally low backgrounds using low cell numbers and even single cells. Beginning with live cells, Cleavage Under Targets and Tagmentation (CUT&Tag) provides amplified sequence-ready libraries in a day on the bench top or in a high-throughput format. Tethering in situ followed by activation of pA-Tn5 results in factor-targeted tagmentation, generating fragments ready for PCR enrichment and DNA sequencing. Here we overcome the limitations of ChIP-seq and CUT&RUN using a transposome that consists of a hyperactive Tn5 transposase 14, 15-Protein A (pA-Tn5) fusion protein loaded with sequencing adapters. Moreover, the release of MNase-cleaved fragments into the supernatant with CUT&RUN is not well-suited for application to single-cell platforms 12, 13. Although CUT&RUN can generate high-quality data from as few as 100–1000 cells, it must be followed by DNA end polishing and adapter ligation to prepare sequencing libraries, which increases the time, cost and effort of the overall procedure. CUT&RUN provides base-pair resolution of specific chromatin components with background levels that are much lower than with ChIP-seq, dramatically reducing the cost of genome-wide profiling. MNase is activated by addition of calcium, and fragments are released into the supernatant for extraction of DNA, library preparation and paired-end sequencing. For example, CUT&RUN, which is based on Laemmli’s Chromatin ImmunoCleavage (ChIC) strategy 11, maps a chromatin protein by successive binding of a specific antibody, and then tethering a Protein A/Micrococcal Nuclease (pA-MNase) fusion protein in permeabilized cells without cross-linking 9. Alternatives to ChIP include enzyme-tethering methods for unfixed cells, such as DamID 7, ChEC-seq 8, and CUT&RUN 9, 10, where a specific protein of interest is targeted in situ and then profiled genome-wide. Chromatin immunoprecipitation with sequencing (ChIP-seq) and its variations 2, 3, 4, 5 suffer from low signals, high backgrounds and epitope masking due to cross-linking, and low yields require large numbers of cells 2, 6. ![]() #Kaya peak insidia full#The advent of massively parallel sequencing and the dramatic reduction in cost per base has fueled a genomics revolution, however, the full promise of epigenomic profiling has lagged owing to limitations in methodologies used for mapping chromatin fragments to the genome 1. We demonstrate the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Here we describe Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. A complete understanding of gene regulation will require the mapping of specific chromatin features in small samples of cells at high resolution. Many chromatin features play critical roles in regulating gene expression. ![]()
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |