DNeasy Blood & Tissue Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
		- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Downstream tips
- Include RNAse treatment for 15-20 min. - Ensure EtOH is completely evaporated off of the column prior to elution. Adjust time from 1min to 5 min at 60`C - Use prewarmed TE buffer to elute the DNA

Publication protocol

MBD-seq and capture verification
Genome-wide DNA methylation patterns were quantified using MBD protein capture (MBD-capture; MethylMiner Kit, Invitrogen), followed by next-generation sequencing. DNA from unstimulated neuronal cultures was extracted, RNase treated and purified (DNeasy, Qiagen). 2 μg of genomic DNA was sonicated to 200–400 bp (Bioruptor Pico, Diagenode). Methylated DNA was collected with recombinant MBD2 protein/biotin complex, which was purified using streptavidin-coated magnetic beads (Invitrogen). DNA sequencing was performed at Hudson Alpha using NEBNext reagents (New England Biolabs) according to manufacturer's recommendations with minor modifications (including the use of custom library adaptors and indexes). DNA libraries were quantified with the Kapa Library Quant Kit (Kapa Biosystems), and underwent sequencing (25 M total 50 bp single-end reads) on an Illumina sequencing platform (HiSeq2000). We sequenced two biological replicates as well as an input (non-IP) control for normalization. To ensure that MBD-capture resulted in the specific enrichment of methylated DNA, we performed control reactions in which gDNA was spiked with synthetic methylated and non-methylated DNA fragments (1 pg each, Methyl Miner kit, Invitrogen) before immunoprecipitation with recombinant MBD2. Methylated and non-methylated DNA capture was quantified via RT-qPCR with primers specific for each synthetic sequence, using both the captured (MBD2-bound) and unbound fractions. Our results demonstrated robust enrichment (∼600-fold) of methylated DNA fragments in the captured sample, and equally robust depletion of methylated DNA in the unbound fraction (Supplementary Fig. 2b).

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Manufacturer protocol

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