Methylminer methylated dna enrichment kit

One μg of genomic DNA from each of the six samples (3 ovaries and 3 hypothalami) was sonicated to produce DNA fragments of about 350 bp. Methyl-binding domain (MBD) enrichment was performed using the MethylMiner™ Methylated DNA Enrichment Kit (Invitrogen, Carlsbad, CA, USA), following manufacture instruction. Sequencing library construction was performed using the TruSeq® Nano Library Preparation Kit (Illumina, San Diego, CA, USA). Libraries were quality checked and quantified on Agilent 2200 TapeStation, High Sensitivity D1000. The six samples were then used for cluster generation and subsequent sequencing on a single lane of Illumina Hiseq 2000 (San Diego, CA) and 100-base paired-end reads were generated.

Bone marrow buffy coats were collected from the patients; the median lymphoid cell percentage was 85.75% (range, 41.60–99.00%). CD19-positive B cells were collected from five healthy donors using magnetic bead sorting (EasySep^TM; STEMCELL Technologies, Inc., Vancouver, Canada). Purity was confirmed using flow cytometry analysis (>95.0%). Genomic DNA was isolated using the Promega Maxwell® 16 MDx Instrument. Genomic DNA (1 µg) was sheared to 200–400 bp using a Covaris LE220 sonicator; the fragments were then subject to methyl-CpG enrichment using the Invitrogen MethylMiner Methylated DNA Enrichment Kit, which uses a recombinant form of human MBD protein-2. The enriched methylated DNA fragments were eluted as a single enriched population with a 2,000 mM NaCl elution buffer. The eluted DNA was then used to generate libraries according to the standard Illumina protocol. Briefly, the DNA fragments were subject to end repair, A-tailing of the 3′ end, Illumina adapter ligation, size selection (aiming for 300–500 bp), PCR amplification, and validation using an Agilent Bioanalyzer. The libraries were then sequenced on the Illumina HiSeq 2000 platforms.

Library construction, bisulfite conversion and sequencing were performed at the Beijing Genomics Institute. Briefly, DNA was fragmented into 100–300 bp fragments by sonication (Covaris S-2, Woburn, MA, USA). The fragmentation parameters were as follows: duty cycle, 10%; intensity, 5; cycles/burst, 200; cycles, 16; total fragmentation time, 960 s. Fragmentation was confirmed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Fragments were end repaired (Illumina) as recommended by the manufacturer. Repaired fragments were ligated with methylated sequencing adaptors using a paired end adaptor oligo kit and oligo mix 5 (Illumina). Ligated fragments were selected by gel electrophoresis and fragments of size 360 bp extracted using a QIAquick gel extraction kit (Qiagen). Size-selected fragments were exposed to a MethylMiner methylated DNA enrichment kit (Invitrogen) and then subsequently bisulfite treated using an EZ DNA methylation kit (Zymo Research, Irvine, CA, USA). Libraries were amplified using T4 polymerase (Enzymatics), and sequenced on Illumina’s HiSeq PE 150 platform.

LCM was used to isolate NM, IM, and GT cells, and cellular DNA was purified and then fragmented to 100-500 bp with gas at 44 psi for 1 min through a nebulizer (Agilent Technologies, Santa Clara, CA) and then enriched for methylated DNA using the MethylMiner Methylated DNA Enrichment kit (Invitrogen). Briefly, methylated DNAs were precipitated from 500 ng fragmented DNAs via binding to the methyl-CpG binding domain of human MBD2, which was coupled to magnetic Dynabeads. The methylated fragments were then eluted with High-Salt Elution Buffer (Invitrogen) and purified using the MinElute PCR Purification kit (Qiagen). The methylated DNA fragments were ligated to one pair of adaptors (S1 Table) for sequencing, size-fractioned on a 2% agarose gel to yield 200- to 300-bp fragments, and subjected to 18 cycles of PCR using primers described in Table S1. Each library was diluted to 8 pM for 76 cycles of single-read sequencing on the Illumina Genome Analyzer II.

For comparing data obtained by different methylation kits, we captured methylated DNA from IMR-90 and SssI DNA as follows. Genomic DNA was sheared to 150 to 200 bp using the Covaris S220 sonicator. MBDs were captured using the MethylMiner Methylated DNA Enrichment Kit (Invitrogen, Carlsbad, USA) and the MethylCap Kit (Diagenode, Liege, Belgium) following the manufacturers’ recommended protocols. The bound fractions were eluted at 500 mM and 1 M NaCl for MethylMiner and with buffers at different salt concentrations (low, medium, high) for MethylCap. Sequencing libraries were prepared with the SOLiD Fragment Library Construction Kit (Applied Biosystems, Foster City, USA). MeDIP-seq methylation immunocapture and library preparation were performed using the MeDIP Kit (Active Motif, Carlsbad, USA) following the manufacturer’s recommended protocol.