CUTANA™ CUT&RUN Library Prep Kit

Gene browser shots generated using the Integrative Genomics Viewer (IGV, Broad Institute) show a representative 137 kb window for H3K27me3 antibody (ABclonal A16199) tested in CUT&RUN using 500k K562 cells. Decreasing inputs of CUT&RUN enriched DNA were used in the library prep protocol to simulate low abundance targets or low cell input experiments. Data are largely indistinguishable across inputs, demonstrating robust preparation of Illumina^® NGS-ready libraries.

CUT&RUN was performed using the CUTANA ChIC/CUT&RUN Kit (EpiCypher 14-1048) starting with 500k K562 cells. Five nanograms of CUT&RUN output DNA from reactions utilizing IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0041), H3K27me3 (ABclonal A16199), and CTCF (EpiCypher 13-2014) antibodies were prepared for paired-end Illumina^® sequencing with the CUTANA CUT&RUN Library Prep Kit. Library DNA was analyzed by Agilent TapeStation^®, which confirmed that mononucleosomes were predominantly enriched in CUT&RUN (~300 bp peaks represent 150 bp nucleosomes + sequencing adapters). Peaks at ~380 bp correspond to the SNAP-CUTANA™ K-MetStat Panel of spike-in controls (EpiCypher 19-1002).

CUT&RUN sequencing libraries described in Figure 2 were sequenced on an Illumina^® NextSeq 2000 with a P3 cartridge (paired-end 2×50 cycle). Data were aligned to the hg19 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and blacklist regions. A representative 640 kb window at the SEPTIN5 gene is shown for three replicates (“Rep”) of IgG and H3K4me3 antibodies, as well as individual tracks for H3K27me3 and the transcription factor CTCF, demonstrating the robustness and reproducibility of the workflow with a variety of targets. Sequencing libraries prepared with the CUTANA CUT&RUN Library Prep kit produced the expected genomic distribution for each target. Sample sequencing depth was as follows (millions of reads): IgG Rep 1 (20.1), IgG Rep 2 (16.6), IgG Rep 3 (16.1), H3K4me3 Rep 1 (7.3), H3K4me3 Rep 2 (17.2), H3K4me3 Rep 3 (13.8), H3K27me3 (13.4), CTCF (16.2). Images were generated using the Integrative Genomics Viewer (IGV, Broad Institute).