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).
- 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).