Hydroxymethyl Collector™ Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Kit is optimized for use with 5-25 ng fragmented (>500bp) DNA per reactions	After restriction digest, samples should be treated for 20 mins at 65'C

Upstream tips
Kit is optimized for use with 5-25 ng fragmented (>500bp) DNA per reactions
Protocol tips
After restriction digest, samples should be treated for 20 mins at 65'C

Publication protocol

Deep-Sequencing Determination of 5meCs and 5hmeCs.
Genome-wide gDNA methylation and hydroxymethylation profiles of mouse iPSCs, EpiLCs, PGCLCs, and E12.5 embryonic PGCs were determined by deep sequencing of 5meC- or 5hmeC-enriched DNA fragments. Genomic DNA was sonicated to 200–300 bp fragments using a Covaris S2 sonicator in AFA microtubes (10–200 ng DNA in 80 μL 0.1× Tris/HCl-EDTA buffer) at 4 °C, and DNA size distribution was determined using Bioanalyzer or Tapestation. DNA fragments were subjected to enrichment for 5meCs and 5hmeCs using biotin-conjugated methyl-CpG–binding domain (MBD) of the recombinant human methyl-CpG–binding domain protein 2 (MBD2) [MBD-sEq. (21)] and selective labeling of 5hmeCs with a biotinylated tag by two-step chemical reactions involving β-glucosyltransferase (23), respectively. Reagents and protocols for enrichment of 5meC- and 5hmeC-rich gDNA fragments were provided in the MethylMiner kit (Thermo Fisher) or the Hydroxymethyl Collector kit (Active Motif), respectively. To avoid sensitivity bias of enrichment reactions resulting from different amounts of input DNA, an equal amount (10 ng) of fragmented DNA was put into each enrichment reaction for both 5meCs and 5hmeCs for all cell types, and eluted DNA from multiple (typically 8–24) 10-ng scale reactions was pooled for ethanol precipitation to yield at least 0.5 ng 5meC-enriched gDNA fragments. For both the 5meC and 5hmeC enrichment kits, the manufacturer-recommended minimum amount of input DNA was 5 ng. Ten-nanogram input DNA has been shown to have sufficient molecular complexity for genome-wide ChIP-seq profiling of the mouse genome for common histone modifications (37). Successful enrichment was monitored by simultaneously performing positive control reactions using kit-provided reagents. The 5meC-rich DNA fragments were eluted in 200 μL per reaction with 2 M NaCl and then desalted/concentrated to 20 μL by ethanol precipitation with Pellet Paint coprecipitant fluorescence dye polymer (Millipore). The 5hmeC-rich DNA fragments were eluted in 50 μL per reaction elution buffer provided in the kit and subjected to ethanol precipitation.

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Manufacturer protocol

Download the product protocol from Active Motif for Hydroxymethyl Collector™ Kit below.

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