GSM2486685: S_MeDIP_1r; Homo sapiens; MeDIP-Seq
Human brain tissue was homogenized in Buffer RTL (Qiagen, # 79216) with a 1 mL syringe fitted with a 20G needle. Genomic DNA was extracted from the homogenate using the AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, # 80224). Bisulfite sequencing DNA fragmentation Genomic DNA was sonicated to 550 bp using a Covaris S2 ultra-sonicator (1 cycle, 2 cycles per burst, 45 seconds, 10% duty cycle, intensity 2.0). Multiple sonications were performed for each sample (2 μg DNA maximum per 100 μL fragmentation). The fragmented aliquots from each sample were pooled together to meet the input needs of both WGB and TAB, prior to any downstream assays. Bisulfite and TET1 treatment For whole genome bisulfite (WGB) sequencing the fragmented DNA was bisulfite treated using reduced conversion times with the EZ DNA Methylation-Lightning™ Kit (Zymo Research) to convert non-methylated cytosines to uracil. For Tet-assisted bisulfite (TAB(44)) sequencing we used the WiseGene kit to treat the fragmented DNA with T4 β-glucosyltransferase (16 hours at 30°C) to protect hmCs from oxidation by installation of a glucose moiety. Treatment with TET1 then oxidized only mCs to 5-carboxylcytosine. Finally, the TET1-oxidized DNA is bisulfite treated using the EZ DNA Methylation-Lightning™ Kit (Zymo Research) to convert non-hydroxymethylated cytosines to uracil. To assess conversion and protection rates, fragmented genomic DNA was spiked with commercial methylated and hydroxymethylated non-mammalian control DNAs (WiseGene). The mC control consisted of SssI treated lambda DNA, which should theoretically contain 100% methylated CpGs and non-methylated non-CpG cytosines. In reality, we observed a 96.3% methylation level for CpGs in the mC control, and likely the result of incomplete methylation during the production of the control. The hmC control consisted of a 1.64 kb amplicon from pUC19, intended to contain 100% hydroxymethylcytosines at all locations. The observed hydroxymethylation level for the hmC control was also reduced at 91.1%, but is not unexpected considering past evidence of contamination of commercial 5-hmCTP with dCTP(45-46). Conversion and protection rates are presented in Supplementary Table 1. Sequencing We used the Accel-NGS™ Methyl-Seq DNA Library Kit (Swift Biosciences) to create indexed Illumina libraries directly from the bisulfite and TET1-oxidized/bisulfite converted DNA.  Libraries were pooled with PhiX (15%) to compensate for lowered cytosine signal and 2x150 bp paired-end sequenced on an Illumina NextSeq 500 using v2 chemistry. Enrichment panel DNA fragmentation Genomic DNA was sonicated to 100-150 bp using a Covaris S2 ultra-sonicator (10 cycles, 1 cycle per burst, 60 seconds, 10% duty cycle, intensity 5.0). Multiple sonications were performed for each sample (2 μg DNA maximum per 100 μL fragmentation). The fragmented aliquots from each sample were pooled together prior to any downstream assays. MBD We used the MethylMiner™ Kit (Invitrogen) to enrich for mCG via affinity purification with a methyl-CG binding domain protein (MBD2). For each sample, 15 μL of stock Dynabeads ® M-280 Streptavidin (10 μL of beads per 1 μg of DNA input) was coupled to 5.25 μg of MBD-Biotin Protein according to the vendor supplied protocol. Prepared MBD-beads were added to each input sample of 1.5 μg of fragmented DNA, and adjusted to a final volume of 200 μL in 1x Bind/Wash Buffer. The MBD-beads were incubated with DNA on an orbital shaker at 650 rpm for 1 hour at room temperature in a 96-well 1.2 mL square well plate. Following incubation the plate was placed on a magnetic rack and the supernatant containing non-captured, mCG-depleted DNA was retained for MBD-DIP. Beads were then washed three times by addition of 200 μL of 1x Bind/Wash Buffer followed by 3 minute incubations at room temperature on an orbital shaker at 650 rpm. Methylated DNA was eluted from the beads with three treatments with 200 μL low salt elution buffer (12.5% High Salt Elution Buffer + 87.5% Low Salt Elution Buffer [v/v]; 500 mM NaCl final), following the same incubation paradigm as the wash steps. The combined 600 μL mCG-enriched eluate was purified by ethanol precipitation. After optimization, we have empirically determined that a low salt elution improves the specificity of the assay for loci with moderate numbers of CpG sites, giving better methylome-wide representation(41). Additionally, shearing genomic DNA to as small fragments as the downstream sequencing protocol allows (100-150 bp) further increases both the resolution and sensitivity of the MBD enrichment assay (i.e. small, sparsely methylated fragments require less energy to be captured than larger fragments). MBD-DIP The mCG-depleted DNA fraction was retained after MBD enrichment and purified by ethanol precipitation. Methylated DNA immunoprecipitation (MeDIP) was used to enrich for mCH from the mCG-depleted fraction of each sample. We used the MagMeDIP kit (Diagenode, # C02010021) with 1.0 μg of mCG-depleted DNA as input. Briefly, input DNA was heat denatured and incubated with anti-mC antibody and magnetic beads overnight at 4°C with end-over-end rotation. After incubation, beads were washed and mCH-enriched DNA liberated with proteinase K treatment. Reagent volumes and concentrations were prepared according to the vendor supplied protocol. The crude mCH enriched fractions were cleaned by column purification (ChIP DNA Clean & Concentrator, Zymo, # D5201) prior to library generation. hMe-Seal Selective chemical labeling and enrichment of hmC (hMe-Seal)(37) was performed using components of the Hydroxymethyl Collector™ kit (Active Motif). We substituted the enzyme in the kit with T4 β-glucosyltransferase from New England Biosciences (# M0357) to improve labeling performance. In our experience, using high-quality and high-activity enzyme is critical for successful hmC enrichment. 1.5 μg of 150bp fragmented gDNA was used as input for each assay. Briefly, T4 β-glucosyltransferase (10 U per sample, 16 hour incubation at 30°C) was used to selectively label hmC residues with 150 μM final UDP-azide-glucose. Each azo-glucosylated hmC was next biotinylated via dibenzocyclooctyl click chemistry by addition of the provided Biotin Conjugate Solution (likely DBCO-S-S-PEG3-Biotin at 150 μM final) with a 2 hour incubation at 37°C. After biotinylation, the labeled DNA was column purified, enriched with paramagnetic streptavidin beads, washed, and eluted following the vendor supplied protocol, with the substitution of end-over-end rotation with agitation on an orbital shaker at 700 rpm in a 96-well 1.2 mL square well plate. In an independent study(38) this chemical capture-based approach compared favorably to hmC enrichment methods using proteins or antibodies. MeDIP MeDIP was performed with 1.0 μg fragmented gDNA using the MagMeDIP Kit (Diagenode, # C02010021). Capture protocol, purification, library preparation, and sequencing were carried out identically to that of the MBD-DIP samples. Sequencing The MBD and hMe-Seal enriched fractions are used to create indexed sequencing libraries using the TruSeq Nano DNA HT Library Prep Kit (Illumina). For MBD-DIP and MeDIP enriched fractions, the Accel-NGS Methyl-Seq DNA Library Kit (Swift Biosciences) was used to generate indexed libraries directly from single-stranded DNA. Libraries were size-selected using SPRI beads to obtain a mean insert size of 150 bp. Libraries were pooled and 75-cycle single-end sequenced on an Illumina NextSeq 500 using v2 chemistry.