CUTANA™ Fluorescent pAG-Tn5 for CUT&Tag

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Figure 2: Size distribution of released chromatin
CUT&Tag was performed as described above. Recovered DNA was directly PCR amplified to produce sequenceready libraries. Agilent TapeStation traces for libraries derived from negative control Rabbit IgG (top), H3K4me3 (middle), and H3K27me3 (bottom) are shown. Excised DNA is highly enriched for mononucleosomes (peak at ~300 bp reflects ~150 bp insert size).

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Figure 3: CUT&Tag data
CUT&Tag was performed as described above. Representative sequencing tracks obtained using CUTANA Fluorescent pAG-Tn5 for CUT&Tag show a 337 kb view of the TRMT2A gene. CUTANA pAG-Tn5 produced clear peaks with genomic distribution consistent with the known biological functions of H3K4me3 and H3K27me3 as well as minimal background in the IgG negative control.

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Figure 4: Protein gel data
CUTANA pAG-Tn5 (1 µg) was resolved via SDS-PAGE and stained with Coomassie blue. The migration and molecular weight of the protein standards are indicated. Uncharged pAG-Tn5 monomer is 78.5 kDa, however once charged with DNA, Tn5 dimerizes to a final complex weight of 192 kDa.

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CUT&Tag methods
CUT&Tag was performed on 100k K562 cells with 0.5 µg of either IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0041), or H3K27me3 (ThermoFisher MA5-11198) antibodies using CUTANA™ Fluorescent pAG-Tn5 (50 nM final; see Technical Datasheet for lot-specific recommendation) following the EpiCypher Direct-to-PCR CUT&Tag protocol. Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 0.5 million reads (IgG), 2.5 million reads (H3K4me3), and 6.3 million reads (H3K27me3). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.