iDeal CUT&Tag kit for Histones
Compatible with histones and some non-histone proteins

The iDeal CUT&Tag protocol involves the binding of cells on a solid phase ConA magnetic beads, allowing magnetic handling of the cells for the major steps of the protocol. Bead-bound cells are permeabilized, incubated with primary antibody against a target of interest and secondary antibody. Then, Diagenode’s protein pA-Tn5 Transposase - loaded is bound to the complex. Protein A guides Tn5 transposase on chromatin to the antibody attached to its target. Tn5 transposase is activated by Mg+2 ions to insert the sequencing adaptors into genomic regions of interest. DNA is then purified and the tagmented genomic regions of interest are amplified by PCR using Diagenode’s Primer Indexes for tagmented libraries.

Successful CUT&Tag results showing a low background with high region-specific enrichment are presented below. Chromatin profiling has been performed on 50,000 K562 cells, using the Diagenode’s iDeal CUT&Tag kit for Histones (Cat. No. C01070020), the Antibody Package for CUT&Tag anti rabbit (Cat. No. C01070022), the 24 UDI for Tagmented Libraries (Cat.No. C01011034) and H3K4me3 (Cat. No. C15410003), H3K27me3 (Cat. No. C15410069) or H3H9me3 antibodies (Cat. No., C15410193) as indicated. The libraries were sequenced on Illumina’s NovaSeq6000 in 2x50 bp mode.

Figure 1. Typical library profiles (Agilent Fragment traces) generated by the iDeal CUT&Tag protocol using 50,000 K562 cells and H3K27me3 (top), H3K4me4 (middle) primary antibodies and IgG control (bottom). Sharp peak at around 40 bp is an excess of free oligonucleotide used for pA-Tn5 loading.

Figure 3. Sequencing profiles of the CUT&Tag libraries. Integrative genomics viewer (IGV) visualization of CUT&Tag experiments using 50,000 of K562 cells and H3K4me3 (blue), H3K27me3 (red) or H3K9me3 (green).

Figure 4. Sequencing profiles of the CUT&Tag libraries. Integrative genomics viewer (IGV) visualization of CUT&Tag experiments using 50,000 (blue) or 10,000 (red)) of K562 cells and H3K27me3 antibody.

Diagenode's iDeal CUT&Tag Kit for Histones, developed for an efficient chromatin profiling on histone marks, can be used for profiling of some transcription factors and co-factors using a mild fixation as described in the manual. 

Successful CUT&Tag results using CTCF and Suz12 antibodies and iDeal CUT&Tag Kit for Histones. 

Chromatin profiling of Suz12 has been performed on 50,000 of fixed Mouse ES-E14TG2a cells (kindly provided by Luciano Di Croce, CRG, Spain) accordingly to the protocol of Diagenode’s iDeal CUT&Tag kit for Histones (Cat. No. C01070020).   Antibody Package for CUT&Tag anti rabbit (Cat. No. C01070022) and the 24 UDI for Tagmented Libraries (Cat. No. C01011034) were used. The libraries were sized using Fragment Analyzer (Agilent) (triplicates, Figure 1, top). Relative enrichment has been confirmed by qPCR using know positive (T_Bra)  and negative (Actb)  loci  (Figure 1, bottom). Libraries were sequenced on Illumina’s NovaSeq6000 in 2x50 bp mode.

Figure 1. Typical library profiles (top) and relative enrichment (bottom) generated by the iDeal CUT&Tag protocol using 50,000 ES-E14TG2a cells and Suz12 Antibody and IgG control (bottom). Sharp peak at around 40 bp is an excess of free oligonucleotide used for pA-Tn5 loading which does not interfere with the sequencing.

Pictures of representative loci were taken on Integrative Genome Viewer (IGV) for CTCF demonstrating the presence of high signal at promoter regions.

Pictures of representative loci were taken on Integrative Genome Viewer (IGV) for SUZ12 and H3K27me3 triplicate samples, showing an overlap between the signal from both proteins, as part of the Polycomb Repressive Complex.

Figure 2. IGV snapshots

Figure 4. CTCF motif
Presence of specific transcription factor binding motifs in the regions identified as CTCF peaks. The top3 motifs are corresponding to the CTCF binding motif.