CUTANA™ Fragmented Controls for DNA Methylation Sequencing
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Powered by BiozThe CUTANA™ Fragmented Controls for DNA Methylation Sequencing set includes pre-fragmented methylated pUC19 and unmethylated Lambda DNA controls optimized for assessing cytosine conversion efficiency in Enzymatic Methyl-seq (NEB® EM-seq™) when performed downstream of meCUT&RUN and Multiomic CUT&RUN workflows (CUT&RUN-EM). EM-seq is the preferred method for achieving base-pair resolution of 5-methylcytosine (5mC) from CUT&RUN DNA libraries. In traditional EM-seq, cytosine conversion controls are fragmented through sonication after mixing with genomic DNA. When CUT&RUN is used prior to EM-seq to excise chromatin regions of interest, pre-fragmented pUC19 and Lambda controls are required.
CUTANA Fragmented Controls for DNA Methylation Sequencing provides a reliable and easy-to-use solution for validating conversion rates and optimizing methylation sequencing protocols in cutting-edge epigenomics experiments.
Figure 1: Control DNA fragment size
Figure 2: Representative EM-seq conversion efficiency
Figure 3: CUT&RUN-EM methods
CUT&RUN-EM was performed using CUTANA™ Fragmented Controls for DNA Methylation Sequencing and the CUTANA™ ChIC/CUT&RUN Kit (EpiCypher 14-1048) starting with 500k K562 cells and 0.5 µg of IgG (EpiCypher 13-0042), H3K4me3 (EpiCypher 13-0060), or H3K36me3 (EpiCypher 13-0058) antibodies. Library preparation was performed with 1 ng of DNA using the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB E8015). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2×50 bp). Sample sequencing depth was 31.5 million reads (IgG), 21.3 million reads (H3K4me3), and 20.6 million reads (H3K36me3). Data were aligned to the hg38 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions. Validation data are representative where noted.
| Item | Cat. No. | QTY |
|---|---|---|
| CUTANA™ CpG Methylated pUC19 Fragmented Control DNA | 18-8001-05 | 20 µL |
| CUTANA™ CpG Unmethylated Lambda Fragmented Control DNA | 18-8002-05 | 20 µL |
| CUTANA™ 0.1X TE Buffer | 21-1025-05 | 2 x 2 mL |
| APPLICATION | ITEM | CAT. NO. |
|---|---|---|
| meCUT&RUN | CUTANA™ meCUT&RUN Kit for DNA Methylation Sequencing | 14-1060-24 |
| Multiomic CUT&RUN | CUTANA™ Multiomic CUT&RUN Controls Set | 14-1802 |
| Multiomic CUT&RUN | CUTANA™ ChIC/CUT&RUN Kit | 14-1048/14-1048-24 |
Perform meCUT&RUN (EpiCypher 14-1060-24) or Multiomic CUT&RUN (EpiCypher 14-1802). Follow instructions for adding CUTANA Fragmented Controls as outlined in each respective user manual (linked below under Documents & Resources). In brief:
- Transfer 1 ng CUT&RUN DNA to a new tube and adjust final volume to 49 µL with 0.1X TE Buffer. If CUT&RUN yields are < 1 ng, use the total amount of recovered DNA.
- In a fresh tube, combine 1 µL Methylated pUC19 DNA and 1 µL Unmethylated Lambda DNA with 98 µL 0.1X TE Buffer.
- Add 1 µL of the combined diluted Fragmented Control DNAs to the 49 µL of CUT&RUN DNA.
This diluted DNA will be the input for EM-seq conversion and library prep using the NEBNext® Enzymatic Methyl-seq v2 Kit (NEB E8015). Follow the EM-seq protocol adjustments as outlined in the meCUT&RUN or Multiomic CUT&RUN user manuals.