Library Prep and Next-Generation Sequencing
Here, learn how to go from CUT&RUN-enriched DNA to sequencing. For a streamlined workflow use our CUTANA™ CUT&RUN Library Prep Kit, optimized specifically for CUT&RUN DNA yields.
Skip to a specific section:
- Section VIII: Next-generation sequencing library prep
- Section IX: Analysis of library fragment size
- Section X: Illumina® sequencing
Section VIII: Next-generation sequencing library prep (~4 hrs)
Notes on library prep
- For some cell types/targets, low CUT&RUN DNA yields are unavoidable. To optimize library prep for low DNA inputs, see this article.
- Do NOT shear or fragment DNA before library prep. Our PCR conditions for library prep specifically amplify DNA fragments from 200 to 700 bp, which eliminates large fragments.
- Prepare Illumina® sequencing libraries using ~5 ng purified CUT&RUN DNA and the CUTANA CUT&RUN Library Prep Kit (EpiCypher 14-1001 & 14-1002).
- For low-abundance targets or if yields <5 ng, use total amount of recovered DNA. Note that IgG and H3K4me3 control antibodies often generate low yields.
- If using other library prep kits, follow EpiCypher's recommended PCR parameters for indexing PCR and library amplification (below). These conditions are specifically optimized for small CUT&RUN fragments (200-700 bp).
|1||98°C||45 sec||1||Hot start activation of DNA Polymerase|
|2||98°C||15 sec||DNA melting|
|3||60°C||10 sec||Hybrid annealing/extension|
|4||Repeat steps 2 and 3 14 times||Amplification|
|5||72°C||60 sec||1||Final extension|
Section IX: Analysis of library fragment size (~1 hr)
Notes on expected yields and fragment size enrichment
- 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). Fragment distributions for positive (e.g. H3K4me3) and negative (e.g. IgG) control reactions can be used to assess yields and validate library prep workflows.
- Final CUT&RUN library concentration is usually 100-200 nM. Libraries ≥1 nM allow pooling at standard concentrations for sequencing, but good data are obtained down to 0.5 nM. If library concentrations are <0.5 nM, read these tips.
- Adapter dimer contamination appears as a peak at ~125 bp and is caused by low input; see this article and the CUTANA CUT&RUN Library Prep Kit Manual for details.
- See this article for troubleshooting low library yields and/or fragment distribution results.
- Use 1 μL purified CUT&RUN library for quantification. Use the Qubit fluorometer with the 1X dsDNA HS Assay Kit per the manufacturer’s instructions.
- For each library, prepare 5 µL at 10 ng/µL for loading onto the Bioanalyzer orTapeStation system. Record the dilution factor, which is needed to calculate library molarity from the results (reported as DNA concentrations in nM for the desired 200 - 700 bp region).
- Load and analyze 1 µL diluted sequencing library using the High Sensitivity DNA Kit (Bioanalyzer) or the D1000 ScreenTape System & Reagents (TapeStation) as per the manufacturer’s instructions.
- The final traces should show predominant enrichment of mononucleosome-sized fragments, such as those yielded by H3K4me3 and CTCF antibodies in Figure 1 (~300 bp: ~170 bp + 125 bp sequencing adapters). Adapter dimers, if present, are observed as a peak at ~125 bp, see this article for more.
Safe pause point. Libraries can be stored at -20˚C for future processing.
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.
Section X: Illumina sequencing
Tips for sequencing CUT&RUN libraries
- Only 3-8 million uniquely aligned reads are needed for adequate CUT&RUN coverage.
- Paired-end sequencing (2 x 50 bp cycles minimum) is recommended for CUT&RUN to enable detection of K-MetStat Panel barcodes.
- See this article for considerations when sequencing low-concentration libraries.
- See this article for basic information on CUT&RUN sequencing analysis.
- Select appropriate Illumina sequencing platform based on the number of CUT&RUN libraries and desired sequencing depth.
- Pool libraries at desired ratios using the molarity calculations from Section IX (200-700 bp region) and load onto Illumina sequencer. General steps:
- Confirm that each library in a multiplexed sequencing run has a unique pair of i5 & i7 indexes. Libraries with the same pair of indexes must be sequenced in separate lanes/flow cells.
- Dilute each library to the same nM concentration, depending on final yields. For NextSeq 2000 and NextSeq 500/550, dilute to 1-4 nM.
- Pool equimolar libraries into one tube.
- Dilute pooled libraries to appropriate concentration and in the volume required for Illumina platform. Follow guidelines from specific Illumina kit to load onto sequencer (support.illumina.com).
- When setting up the sequencing run, make sure dual i5 & i7 indexes are correctly assigned for each library.
- For H3K4me3 and IgG control reactions spiked with the K-MetStat Panel, align paired-end sequencing reads to the PTM-specific DNA barcodes. Use this data to validate your workflow, identify failed reactions, and troubleshoot problematic experiments. See this article for guidance.
- If control reactions generate expected results, proceed to analysis of experimental reactions. Align paired-ends reads to the appropriate reference genome (e.g. using Bowtie 2) as described.
- For sequencing normalization using E. coli Spike-in DNA, see this article.
(1) This Product is covered by one or more patents, trademarks and/or copyrights owned or controlled by NEB. While NEB develops and validates its products for various applications, the use of this product may require the purchaser to obtain additional third-party intellectual property rights for certain applications.
(2) This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.