Confirm quality of sequencing library

Fragment distribution of purified library

  • Fragment distribution analysis of purified sequencing libraries is the single BEST method to confirm CUT&RUN success.
  • Libraries should show enrichment of mononucleosome-sized DNA fragments (~300 bp, including CUT&RUN DNA + sequencing adapters) (see Figure 1 below).

Library yields

  • In general, library yields should not be used to determine assay success. Library yields vary widely by cell type, number of cells, target abundance, and antibody quality.
  • Aim for a library concentration of ≥ 1 nM, which will enable pooling at standard concentrations for multiplexed sequencing.
  • For library concentrations below 0.5 nM, we recommend visiting considerations for library prep and sequencing from low CUT&RUN yields and see additional tips here for experiments that cannot be repeated.

Figure 1. Typical TapeStation traces from CUTANA™ CUT&RUN libraries prepared using antibodies targeting IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0041), and CTCF (EpiCypher 13-2014). All libraries are predominantly enriched for mononucleosome-sized fragments, as indicated by the peak at ~300 bp (~170 bp nucleosomes + sequencing adapters).

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