CUTANA™

CUTANA™ GST-MeCP2 for meCUT&RUN

$245.00
SKU: 15-2002-S
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  • Type:  Reader Protein
  • Mol Wgt:  39.1 kDa
  • Host:  E. coli
  • Epitope Tag:  GST, 6xHis

Description

CUTANA™ GST-MeCP2 for meCUT&RUN is a GST-tagged MeCP2 methyl binding domain that is validated to enrich regions of chromatin with symmetrically methylated CpGs in CUTANA™ meCUT&RUN, a modified CUT&RUN workflow that enables streamlined, high-resolution mapping of DNA methylation. meCUT&RUN can be followed by a traditional library prep method, such as the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002), to provide ~150 bp resolution profiles of DNA methylation enrichment, or by a cytosine conversion strategy such as Enzymatic Methyl-seq (NEB® EM-seq™, preferred) or bisulfite sequencing to provide base-pair resolution of 5-methylcytosine (5mC).

Validation Data

15-2002-dna-fragment-size-distribution-analysis (Click to enlarge)

Figure 1: meCUT&RUN DNA fragment size distribution analysis
meCUT&RUN was performed as described in Figure 3. Library DNA was analyzed by Agilent TapeStation®. This analysis confirmed that mononucleosomes were predominantly enriched in meCUT&RUN (~300 bp peaks represent 150 bp nucleosomes + sequencing adapters).

15-2002-gene-browser-tracks (Click to enlarge)

Figure 2: Gene browser tracks
meCUT&RUN was performed as described in Figure 3. A 30 kb window at the AJM1 gene is shown for anti-GST antibody and meCUT&RUN. Tracks are also shown with representative data for meCUT&RUN followed by EM-seq (meCUT&RUN-EM) and whole genome EM-seq (WGEM), using the New England Biolabs NEBNext® Enzymatic Methyl-seq v2 Kit (NEB E8015). The meCUT&RUN kit produced the expected genomic distribution, showing enrichment of methylated DNA that approximates the methylated CpG pattern observed in WGEM. Images were generated using the Integrative Genomics Viewer (IGV, Broad Institute).

meCut&RUN-methods (Click to enlarge)

Figure 3: meCUT&RUN methods
meCUT&RUN was performed starting with 500k K562 cells with either 2.5 µL of 20X GST-MeCP2 added as the primary binding reagent or 0.5 µg of a secondary antibody-only control (anti-GST antibody, EpiCypher 13-0073) to determine background cleavage. Library preparation was performed using 5 ng of meCUT&RUN-enriched DNA (or the total amount recovered if less than 5 ng) using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001/14-1002). Libraries were run on an Illumina NextSeq2000 with paired-end sequencing (2x50 bp). Sample sequencing depth was 7.7 million reads (anti-GST) and 8.4 million reads (GST-MeCP2). Data were aligned to the hg38 genome using Bowtie2. Data were filtered to remove duplicates, multi-aligned reads, and ENCODE DAC Exclusion List regions.

Recommended Accessory Products

ITEM CAT. NO.
CUTANA™ Anti-GST Tag Antibody 13-0073
CUTANA™ ChIC/CUT&RUN Kit 14-1048/14-1048-24
CUTANA™ CUT&RUN Library Prep Kit 14-1001/14-1002

Technical Information

Storage
Stable for 12 months at -20°C from date of receipt. The protein is not subject to freeze/thaw under these conditions.
Formulation
25 mM HEPES-NaOH pH 6.5, 200 mM NaCl, 1 mM DTT, 50% glycerol, 2 mM MgCl2

Application Notes

To perform meCUT&RUN with this reagent and a CUTANA CUT&RUN Kit (EpiCypher 14-1048), please follow the meCUT&RUN Kit Manual. If using homemade buffers, adjust the EpiCypher CUT&RUN Protocol as follows:

    Day 1:
  • Prepare Pre-Wash Buffer with 200 mM NaCl*. This is used to prepare Wash Buffer, Digitonin Buffer, and Antibody Buffer.
    • *NOTE: Standard CUT&RUN uses 150 nM NaCl in these buffers; however, excess salt is recommended to improve MeCP2 specificity for methylated DNA.
  • In place of a primary antibody, add 2.5 µL of GST-MeCP2 to each reaction, and incubate on a nutator at 4˚C overnight.
    Day 2:
  • On Day 2, discard the GST-MeCP2 supernatant and resuspend each reaction in 50 µL cold Digitonin Buffer.
  • Add 1.0 µL anti-GST Tag Antibody (EpiCypher 13-0073)* to each reaction and incubate on a nutator at room temperature for 30 minutes.
    • *NOTE: Secondary-only (anti-GST Tag) reactions are recommended as a negative control.
  • At the end of the incubation, discard the supernatant and wash two times with 200 µL cold Digitonin Buffer.
  • Resuspend in 50 µL per reaction cold Digitonin Buffer and proceed to pAG-MNase (EpiCypher 15-1016/15-1116) addition per the standard protocol.
    Sequencing Libraries can be prepared using two methods:
  • Option 1: Traditional library prep, which provides a CUT&RUN-like view of genomic regions with high DNA methylation. The CUTANA CUT&RUN Library Prep Kits (EpiCypher 14-1001/14-1002) are compatible with this approach.
  • Option 2: A cytosine conversion strategy (e.g., New England Biolabs NEBNext® Enzymatic Methyl-seq v2 Kit, E8015), which provides base-pair resolution of 5mC enrichment. Prior to EM-seq conversion, add CUTANA™ Fragmented Controls for DNA Methylation Sequencing (EpiCypher 14-1804) to each reaction as outlined in the 14-1804 Technical Data Sheet.
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